Modified therapeutic agents, stapled peptide lipid conjugates, and compositions thereof

ABSTRACT

Methods and compositions are provided for extending the half-life of a therapeutic agent. A modified therapeutic agent (mTA) comprises a therapeutic agent, a staple, and a half-life extending molecule. The mTAs disclosed herein may be used to treat a disease or a condition in a subject in need thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage entry of InternationalApplication No. PCT/US2014/070977, filed Dec. 17, 2017; which claims thebenefit of priority from U.S. Provisional Application No. 61/917,816filed Dec. 18, 2013, which is incorporated by reference herein in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 6, 2016, isnamed 41135-717.831-SL_ST25.txt and is 26,748 bytes in size.

BACKGROUND OF THE INVENTION

The development of therapeutic agents (e.g., biological drugs) is oftenhampered by short half-lives. The biological half-life or eliminationhalf-life of a substance is the time it takes for a substance (forexample a metabolite, drug, signaling molecule, radioactive nuclide, orother substance) to lose half of its pharmacologic, physiologic, orradiologic activity. As a result of the short half-life, patients areoften administered higher dosages more frequently, which may lead toreduced compliance, higher costs and greater risks of side effects.

Extended-release products are designed to prolong the absorption ofdrugs with short half-lives, thereby allowing longer dosing intervalswhile minimizing fluctuations in serum drug levels. Current strategiesused for extending half-lives are those that increase hydrodynamicvolume (PEGylation) or those that use FcRn-mediated recycling (albuminfusions). Attachment of polypeptides or lipophilic constituents to drugshas also been used to extend the half-life of a biological agent (U.S.Pat. No. 6,268,343; U.S. Pat. No. 5,750,497; U.S. Pat. No. 8,129,343).

The present disclosure provides modified therapeutic agents (mTAs) forimproving the biological, chemical, physiologic, pharmacologic,pharmacokinetic, and/or pharmacodynamic properties of a therapeuticagent.

SUMMARY OF THE INVENTION

Disclosed herein are modified therapeutic agents (mTAs) comprising atherapeutic agent, a first staple, and a first half-life extendingmolecule, wherein the therapeutic agent is a modified or unmodifiedtherapeutic peptide that is covalently attached to the first staple viatwo amino acid residues on the modified or unmodified therapeuticpeptide and the half-life of the mTA is longer than the half-life of theunmodified therapeutic peptide alone. The first half-life extendingmolecule may be covalently attached to the first staple. The firsthalf-life extending molecule may comprise a lipid, a polyglycol region,or a combination thereof. The first half-life extending molecule maycomprise a lipid. The first half-life extending molecule may comprise alipid and a polyglycol region. The first half-life extending moleculemay comprise a polyglycol region. The lipid may be selected from a groupconsisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols, and derivatives thereof. The polyglycol region maycomprise one or more polyethylene glycol units, polypropylene glycolunits, or polybutylene glycol units, or a combination thereof. Thepolyglycol region may be selected from

wherein m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20. The modified therapeutic peptide maycomprise one or more amino acid additions, deletions, or substitutions,or a combination thereof. The unmodified therapeutic peptide may beselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, GIP,GLP1R and GCGR dual agonist, GLP1R and GIPR dual agonist, and GLP1R,GCGR and GIPR tri-agonist. The modified therapeutic peptide may be aderivative of a peptide selected from GLP-1, glucagon, oxyntomodulin,exendin-4, GLP-2, GIP, GLP1R and GCGR dual agonist, GLP1R and GIPR dualagonist, and GLP1R, GCGR and GIPR tri-agonist; the derivative being apeptide comprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence comprising atleast a portion of a polypeptide sequence selected from a groupconsisting of selected from a group consisting of SEQ ID NO: 1-53. Themodified or unmodified therapeutic peptide may comprise an amino acidsequence comprising 10 or more amino acids based on or derived from apolypeptide sequence selected from a group consisting of selected from agroup consisting of SEQ ID NO: 1-53. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence that is at leastabout 50% homologous to an amino acid sequence selected from the groupcomprising SEQ ID NO: 1-53. The modified or unmodified therapeuticpeptide may comprise an amino acid sequence that is at least 80%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 1-53. The modified or unmodified therapeutic peptide maycomprise an amino acid sequence comprising at least a portion of apolypeptide sequence selected from a group consisting of selected from agroup consisting of SEQ ID NO: 8-12. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence comprising 10 ormore amino acids based on or derived from a polypeptide sequenceselected from a group consisting of selected from a group consisting ofSEQ ID NO: 8-12. The modified or unmodified therapeutic peptide maycomprise an amino acid sequence that is at least about 50% homologous toan amino acid sequence selected from the group comprising SEQ ID NO:8-12. The modified or unmodified therapeutic peptide may comprise anamino acid sequence that is at least 80% homologous to an amino acidsequence selected from the group comprising SEQ ID NO: 8-12. Themodified or unmodified therapeutic peptide may comprise an amino acidsequence comprising at least a portion of a polypeptide sequenceselected from a group consisting of selected from a group consisting ofSEQ ID NO: 15-19. The modified or unmodified therapeutic peptide maycomprise an amino acid sequence comprising 10 or more amino acids basedon or derived from a polypeptide sequence selected from a groupconsisting of selected from a group consisting of SEQ ID NO: 15-19. Themodified or unmodified therapeutic peptide may comprise an amino acidsequence that is at least about 50% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 15-19. The modified orunmodified therapeutic peptide may comprise an amino acid sequence thatis at least 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 15-19. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence comprising atleast a portion of a polypeptide sequence selected from a groupconsisting of selected from a group consisting of SEQ ID NO: 20-29. Themodified or unmodified therapeutic peptide may comprise an amino acidsequence comprising 10 or more amino acids based on or derived from apolypeptide sequence selected from a group consisting of selected from agroup consisting of SEQ ID NO: 20-29. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence that is at leastabout 50% homologous to an amino acid sequence selected from the groupcomprising SEQ ID NO: 20-29. The modified or unmodified therapeuticpeptide may comprise an amino acid sequence that is at least 80%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 20-29. The modified or unmodified therapeutic peptide maycomprise an amino acid sequence comprising at least a portion of apolypeptide sequence selected from a group consisting of selected from agroup consisting of SEQ ID NO: 30-53. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence comprising 10 ormore amino acids based on or derived from a polypeptide sequenceselected from a group consisting of selected from a group consisting ofSEQ ID NO: 30-53. The modified or unmodified therapeutic peptide maycomprise an amino acid sequence that is at least about 50% homologous toan amino acid sequence selected from the group comprising SEQ ID NO:30-53. The modified or unmodified therapeutic peptide may comprise anamino acid sequence that is at least 80% homologous to an amino acidsequence selected from the group comprising SEQ ID NO: 30-53. Each ofthe two amino acid residues may be cysteine. At least one of the twoamino acid residues may be an amino acid addition or substitution on themodified therapeutic peptide. The two or more residues may be at leastabout 4 amino acid residues apart. The two or more residues may be atleast about 7 amino acid residues apart. The two or more residues may beat least about 11 amino acid residues apart. The mTA may furthercomprise a second staple. The mTA may further comprise a secondhalf-life extending molecule. The mTA may further comprise a secondstaple and a second half-life extending molecule, wherein the secondhalf-life molecule is covalently attached to the second staple.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent, a first staple, and a first half-lifeextending molecule, wherein the therapeutic agent is a modified orunmodified therapeutic peptide that is covalently attached to the firststaple via two amino acid residues on the modified or unmodifiedtherapeutic peptide and the half-life of the mTA is longer than thehalf-life of the unmodified therapeutic peptide alone. The first staplemay comprise:

wherein each X is independently selected from O, NH, and S; each Y isindependently selected from N and CH; each Z is independently selectedfrom N and CH; each R⁸ and R⁹ is independently H or —C(O)(alkyl) orcomprises a linker to the first half-life extending molecule; each R¹⁰and R¹¹ is independently oxo or comprises a linker to the firsthalf-life extending molecule; R¹² is independently hydroxy, alkoxy, orcomprises a linker to the first half-life extending molecule; a is 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17; b is 1, 2, 3, 4,5, or 6; and c is 1 or 2. The first half-life extending molecule maycomprise a lipid, a polyglycol region, or a combination thereof. Thefirst half-life extending molecule may comprise a lipid. The firsthalf-life extending molecule may comprise a lipid and a polyglycolregion. The first half-life extending molecule may comprise a polyglycolregion. The lipid may be selected from a group consisting of sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, fatty amines, and fatty alcohols, andderivatives thereof. The polyglycol region may comprise one or morepolyethylene glycol units, polypropylene glycol units, or polybutyleneglycol units, or a combination thereof. The polyglycol region may beselected from m and

wherein m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20. The modified therapeutic peptide maycomprise one or more amino acid additions, deletions, or substitutions,or a combination thereof. The unmodified therapeutic peptide may beselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, GIP,GLP1R and GCGR dual agonist, GLP1R and GIPR dual agonist, and GLP1R,GCGR and GIPR tri-agonist. The modified therapeutic peptide may be aderivative of a peptide selected from GLP-1, glucagon, oxyntomodulin,exendin-4, GLP-2, GIP, GLP1R and GCGR dual agonist, GLP1R and GIPR dualagonist, and GLP1R, GCGR and GIPR tri-agonist; the derivative being apeptide comprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence comprising atleast a portion of a polypeptide sequence selected from a groupconsisting of selected from a group consisting of SEQ ID NO: 1-53. Themodified or unmodified therapeutic peptide may comprise an amino acidsequence comprising at least a portion of a polypeptide sequenceselected from a group consisting of selected from a group consisting ofSEQ ID NO: 8-12. The modified or unmodified therapeutic peptide maycomprise an amino acid sequence comprising at least a portion of apolypeptide sequence selected from a group consisting of selected from agroup consisting of SEQ ID NO: 15-19. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence comprising atleast a portion of a polypeptide sequence selected from a groupconsisting of selected from a group consisting of SEQ ID NO: 20-29. Themodified or unmodified therapeutic peptide may comprise an amino acidsequence comprising at least a portion of a polypeptide sequenceselected from a group consisting of selected from a group consisting ofSEQ ID NO: 30-53. Each of the two amino acid residues may beindependently selected from cysteine. At least one of the two amino acidresidues may be an amino acid addition or substitution on the modifiedtherapeutic peptide. The two or more residues may be at least about 4amino acid residues apart. The two or more residues may be at leastabout 7 amino acid residues apart. The two or more residues may be atleast about 11 amino acid residues apart. The mTA may further comprise asecond staple. The mTA may further comprise a second half-life extendingmolecule. The mTA may further comprise a second staple and a secondhalf-life extending molecule, wherein the second half-life molecule iscovalently attached to the second staple. The half-life of the mTA maybe 5-fold longer than the half-life of the unmodified therapeuticpeptide alone.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a firsthalf-life extending molecule, wherein the TA is attached to the firststaple. The TA may comprise a modified or unmodified therapeuticpeptide. The modified therapeutic peptide may comprise one or more aminoacid additions, deletions, or substitutions, or a combination thereof.The modified therapeutic peptide may be a derivative of a peptideselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP,GLP1R and GCGR dual agonist, GLP1R and GIPR dual agonist, and GLP1R,GCGR and GIPR tri-agonist; the derivative being a peptide comprising oneor more amino acid additions, deletions, or substitutions, or acombination thereof. The unmodified therapeutic peptide may be selectedfrom GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, GIP, GLP1R andGCGR dual agonist, GLP1R and GIPR dual agonist, and GLP1R, GCGR and GIPRtri-agonist. The modified or unmodified therapeutic peptide may comprisean amino acid sequence comprising at least a portion of a polypeptidesequence selected from a group consisting of selected from a groupconsisting of SEQ ID NO: 1-53. The modified or unmodified therapeuticpeptide may comprise an amino acid sequence comprising 10 or more aminoacids based on or derived from a polypeptide sequence selected from agroup consisting of selected from a group consisting of SEQ ID NO: 1-53.The modified or unmodified therapeutic peptide may comprise an aminoacid sequence that is at least about 50% homologous to an amino acidsequence selected from the group comprising SEQ ID NO: 1-53. Themodified or unmodified therapeutic peptide may comprise an amino acidsequence that is at least 80% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 1-53. The modified orunmodified therapeutic peptide may comprise an amino acid sequencecomprising at least a portion of a polypeptide sequence selected from agroup consisting of selected from a group consisting of SEQ ID NO: 8-12.The modified or unmodified therapeutic peptide may comprise an aminoacid sequence comprising 10 or more amino acids based on or derived froma polypeptide sequence selected from a group consisting of selected froma group consisting of SEQ ID NO: 8-12. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence that is at leastabout 50% homologous to an amino acid sequence selected from the groupcomprising SEQ ID NO: 8-12. The modified or unmodified therapeuticpeptide may comprise an amino acid sequence that is at least 80%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 8-12. The modified or unmodified therapeutic peptide maycomprise an amino acid sequence comprising at least a portion of apolypeptide sequence selected from a group consisting of selected from agroup consisting of SEQ ID NO: 15-19. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence comprising 10 ormore amino acids based on or derived from a polypeptide sequenceselected from a group consisting of selected from a group consisting ofSEQ ID NO: 15-19. The modified or unmodified therapeutic peptide maycomprise an amino acid sequence that is at least about 50% homologous toan amino acid sequence selected from the group comprising SEQ ID NO:15-19. The modified or unmodified therapeutic peptide may comprise anamino acid sequence that is at least 80% homologous to an amino acidsequence selected from the group comprising SEQ ID NO: 15-19. Themodified or unmodified therapeutic peptide may comprise an amino acidsequence comprising at least a portion of a polypeptide sequenceselected from a group consisting of selected from a group consisting ofSEQ ID NO: 20-29. The modified or unmodified therapeutic peptide maycomprise an amino acid sequence comprising 10 or more amino acids basedon or derived from a polypeptide sequence selected from a groupconsisting of selected from a group consisting of SEQ ID NO: 20-29. Themodified or unmodified therapeutic peptide may comprise an amino acidsequence that is at least about 50% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 20-29. The modified orunmodified therapeutic peptide may comprise an amino acid sequence thatis at least 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 20-29. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence comprising atleast a portion of a polypeptide sequence selected from a groupconsisting of selected from a group consisting of SEQ ID NO: 30-53. Themodified or unmodified therapeutic peptide may comprise an amino acidsequence comprising 10 or more amino acids based on or derived from apolypeptide sequence selected from a group consisting of selected from agroup consisting of SEQ ID NO: 30-53. The modified or unmodifiedtherapeutic peptide may comprise an amino acid sequence that is at leastabout 50% homologous to an amino acid sequence selected from the groupcomprising SEQ ID NO: 30-53. The modified or unmodified therapeuticpeptide may comprise an amino acid sequence that is at least 80%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 30-53. The first half-life extending molecule may be attachedto the first staple. The TA may be covalently attached to the firststaple via two amino acid residues in the TA. Each of the two or moreamino acid residues may be cysteine. The one or more of the two or moreamino acid residues may be an amino acid addition or substitution. Thetwo or more residues may be at least about 4 amino acid residues apart.The two or more residues may be at least about 7 amino acid residuesapart. The two or more residues may be at least about 11 amino acidresidues apart. The first half-life extending molecule may comprise alipid, a polyglycol region, or a combination thereof. The firsthalf-life extending molecule may be a lipid. The first half-lifeextending molecule may comprise a lipid and a polyglycol region. Thefirst half-life extending molecule may comprise a polyglycol region. Thelipid may be selected from a group consisting of sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, fatty amines, and fatty alcohols, and derivativesthereof. The polyglycol region may comprise one or more polyethyleneglycol units, polypropylene glycol units, or polybutylene glycol units,or a combination thereof. A half-life of the mTA may be longer than ahalf-life of the TA.

Disclosed herein are compositions comprising the mTAs disclosed herein.

Disclosed herein are methods for treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject a composition comprising a therapeutically effective amount ofan mTA disclosed herein. The disease or condition may be diabetes orobesity, or a medical condition associated with diabetes or obesity. Thedisease or condition may be non-alcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), or cardiovascular disease. Thedisease or condition may be short bowel syndrome (SBS). The disease orcondition may be inflammatory bowel disease (IBD), inflammatory bowelsyndrome (IBS), or psoriasis. The disease or condition may be Crohn'sdisease or ulcerative colitis. The disease or condition may beAlzheimer's disease, Parkinson's disease or Huntington's disease. Themethod may further comprise administering to the subject one or moreadditional therapeutic agents. The one or more additional therapeuticagents may be selected from a group consisting of other diabetes drugs,DPP4 inhibitors, SGLT2 inhibitors, hypoglycemic drugs and biguanidinedrugs, insulin secretogogues and sulfonyl urea drugs, TZD drugs, insulinand insulin analogs, FGF21 and analogs, leptin or leptin analogs, amylinand amylin analogs, an anti-inflammatory drug, cyclosporine A or FK506,5-ASA, and a statin, or any combination thereof.

A modified therapeutic agent may be a peptide lipid conjugate. Disclosedherein are peptide lipid conjugates (PLCs) comprising (a) one or morelipids; and (b) one or more peptide conjugates (PC), the peptideconjugate (PC) comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, wherein the one or more lipids are attached to theone or more peptide conjugates. The two or more residues in the peptideregion may comprise cysteine. The one or more lipids may be attached tothe one or more staples in the peptide region. Alternatively oradditionally, the one or more lipids may be attached to the one or morepeptide therapeutic agents in the peptide region.

Further disclosed herein are peptide lipid conjugates (PLCs) comprising(a) one or more lipids, the lipids selected from a group consisting ofsterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, and fatty alcohols; and (b) one ormore peptide conjugates (PC), the peptide conjugate comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more peptide conjugates. The twoor more residues in the peptide region may comprise cysteine. The one ormore lipids may be attached to the one or more staples in the peptideregion. Alternatively or additionally, the one or more lipids may beattached to the one or more peptide therapeutic agents in the peptideregion.

Further disclosed herein are peptide lipid conjugates (PLCs) comprising(a) one or more lipids; and (b) one or more peptide conjugates (PC),wherein the peptide conjugate comprises a peptide region comprising oneor more peptide therapeutic agents (TA) and a staple region comprisingone or more staples, the one or more staples connect two or moreresidues in the peptide region, the one or more peptide therapeuticagents comprising one or more oxyntomodulin, exenatide (exendin-4),glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, a GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof, wherein the one or more lipids areattached to the one or more peptide conjugates. The two or more residuesin the peptide region may comprise cysteine. The one or more lipids maybe attached to the one or more staples in the peptide region.Alternatively or additionally, the one or more lipids may be attached tothe one or more peptide therapeutic agents in the peptide region.

Disclosed herein are peptide lipid conjugates (PLCs) comprising (a) oneor more lipids; and (b) one or more peptide conjugates (PC), the peptideconjugate (PC) comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, wherein the one or more lipids are attached to theone or more staples. The two or more residues in the peptide region maycomprise cysteine. At least one of the two or more residues may becysteine. The two or more residues may be at least about 4 amino acidresidues apart. The two or more residues may be at least about 7 aminoacid residues apart. The two or more residues may be at least about 11amino acid residues apart.

Further disclosed herein are peptide lipid conjugates (PLCs) comprising(a) one or more lipids, the lipids selected from a group consisting ofsterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, and fatty alcohols; and (b) one ormore peptide conjugates (PC), the peptide conjugate comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more staples. At least one of thetwo or more residues may be cysteine. The two or more residues in thepeptide region may comprise cysteine. The two or more residues may be atleast about 4 amino acid residues apart. The two or more residues may beat least about 7 amino acid residues apart. The two or more residues maybe at least about 11 amino acid residues apart.

Further disclosed herein are peptide lipid conjugates (PLCs) comprising(a) one or more lipids; and (b) one or more peptide conjugates (PC),wherein the peptide conjugate comprises a peptide region comprising oneor more peptide therapeutic agents (TA) and a staple region comprisingone or more staples, the one or more staples connect two or moreresidues in the peptide region, the one or more peptide therapeuticagents comprising one or more oxyntomodulin, exenatide (exendin-4),glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, a GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof, wherein the one or more lipids areattached to the one or more staples. At least one of the two or moreresidues may be cysteine. The two or more residues in the peptide regionmay comprise cysteine. The two or more residues may be at least about 4amino acid residues apart. The two or more residues may be at leastabout 7 amino acid residues apart. The two or more residues may be atleast about 11 amino acid residues apart.

Disclosed herein are peptide lipid conjugates (PLCs) comprising (a) twoor more lipids; and (b) one or more peptide conjugates (PC), the peptideconjugate (PC) comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, wherein at least one of the two or more lipids areattached to the one or more therapeutic agents and at least one of thetwo or more lipids are attached to the one or more staples. The two ormore residues in the peptide region may comprise cysteine.

Further disclosed herein are peptide lipid conjugates (PLCs) comprising(a) two or more lipids, the lipids selected from a group consisting ofsterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, and fatty alcohols; and (b) one ormore peptide conjugates (PC), the peptide conjugate comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein at least oneof the two or more lipids are attached to the one or more therapeuticagents and at least one of the two or more lipids are attached to theone or more staples. The two or more residues in the peptide region maycomprise cysteine.

Further disclosed herein are peptide lipid conjugates (PLCs) comprising(a) two or more lipids; and (b) one or more peptide conjugates (PC),wherein the peptide conjugate comprises a peptide region comprising oneor more peptide therapeutic agents (TA) and a staple region comprisingone or more staples, the one or more staples connect two or moreresidues in the peptide region, the one or more peptide therapeuticagents comprising one or more oxyntomodulin, exenatide (exendin-4),glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, a GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof, wherein at least one of the two ormore lipids are attached to the one or more therapeutic agents and atleast one of the two or more lipids are attached to the one or morestaples. The two or more residues in the peptide region may comprisecysteine.

The PLCs disclosed herein may further comprise one or more polyethyleneglycol subunits.

The PLCs disclosed herein may comprise one or more lipids. The one ormore lipids may comprise one or more sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,or fatty alcohols. The one or more lipids may comprise one or moremyristic acids, docosahexanoic acids, lithocholic acid esters, cholicacids and palmitic acids. The one or more lipids may comprise myristicacid. The one or more lipids may comprise docosahexanoic acid. The oneor more lipids may comprise lithocholic acid ester. The one or morelipids may comprise cholic acid. The one or more lipids may comprisepalmitic acid. The one or more lipids may be pegylated. Alternatively,the one or more lipids are not pegylated.

The one or more lipids may enhance one or more pharmacokineticproperties of the one or more TAs. The one or more lipids may enhanceone or more pharmacokinetic properties of the one or more TAs by atleast about 200% as measured by pharmacodynamics when compared to theone or more TAs not attached to the one or more lipids. The one or morepharmacokinetic properties may comprise a half-life.

The PLCs disclosed herein may comprise one or more peptide conjugates(PCs). The one or more peptide conjugates may comprise one or morepeptide therapeutic agents (TAs). The one or more TAs may comprise atleast a portion of one or more proteins, biomolecules, chemicals,toxins, or drugs, or any combination thereof. The one or more TAs maycomprise at least a portion of one or more hormones, kinases, receptors,ligands, growth factors, regulatory proteins, metabolic proteins,cytokines, or antibodies, or any combination thereof. The growth factormay be a GCSF, GMCSF or FGF21. The GCSF may be a bovine GCSF.Alternatively, the GCSF may be a human GCSF. The GMCSF and/or the FGF21may be from a human. The one or more TAs may be a derived from acytokine. The cytokine may be a beta-interferon. TA may be a derivedfrom a hormone. The hormone may be an exendin-4, GLP-1, somatostatin, orerythropoietin. The GLP-1 and/or erythropoietin may be from a human. Theone or more TAs may be a derived from a toxin. The toxin may be a Mokal,VM-24, ziconotide, chlorotoxin, or protoxin2 (ProTxII). The one or moreTAs may be IL8, ziconotide, somatostatin, chlorotoxin, SDF1(alpha), orIL21.

The one or more TAs may comprise glucagon or derivative thereof. The oneor more TAs may comprise glucagon-like protein-1 (GLP-1) or derivativethereof. The one or more TAs may comprise exenatide or derivativethereof. The one or more TAs may comprise exendin-4 or derivativethereof. The one or more TAs may comprise oxyntomodulin or derivativethereof. The one or more TAs may comprise GLP-2 or derivative thereof.The one or more TAs may comprise a GLP-1R and GIPR dual agonist. The oneor more TAs may comprise a GLP-1R and GCGR dual agonist. The one or moreTAs may comprise a GLP1R, GCGR and GIPR tri-agonist.

The one or more TAs may comprise a polypeptide derivative. Thepolypeptide derivative may comprises at least a portion of a wild-typepolypeptide comprising one or more amino acid mutations. The one or moreamino acid mutations may comprise a deletion, substitution, addition ora combination thereof. The one or more amino acid mutations may compriseadding one or more amino acid residues to a wild-type polypeptide. Theone or more amino acid mutations may comprise deletion of one or moreamino acid residues of the wild-type polypeptide. The one or more aminoacid mutations may comprise substitution of one or more amino acidresidues of the wild-type polypeptide. The one or more amino acidmutations may comprise substituting one or more amino acid residues ofthe wild-type polypeptide with one or more cysteine residues. The one ormore amino acid mutations may comprise substituting one or more aminoacid residues of the wild-type polypeptide with one or more D-amino acidresidues. The one or more amino acid residues of the wild-typepolypeptide may comprise one or more alanines, methionines, arginines,serines, threonines, and tyrosines.

The one or more TAs may comprise at least a portion of a polypeptidesequence selected from a group comprising SEQ ID NO: 1-53. The one ormore TAs may comprise 10 or more amino acids based on or derived from apolypeptide sequence selected from a group comprising SEQ ID NO: 1-53.The one or more TAs may comprise an amino acid sequence that is at leastabout 50% homologous to an amino acid sequence selected from the groupcomprising SEQ ID NO: 1-53. The one or more TAs may comprise an aminoacid sequence that is at least 80% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 1-53. The one or more TAsmay comprise at least a portion of a polypeptide sequence selected froma group comprising SEQ ID NO: 8-12. The one or more TAs may comprise 10or more amino acids based on or derived from a polypeptide sequenceselected from a group comprising SEQ ID NO: 8-12. The one or more TAsmay comprise an amino acid sequence that is at least about 50%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 8-12. The one or more TAs may comprise an amino acid sequencethat is at least 80% homologous to an amino acid sequence selected fromthe group comprising SEQ ID NO: 8-12. The one or more TAs may compriseat least a portion of a polypeptide sequence selected from a groupcomprising SEQ ID NO: 15-19. The one or more TAs may comprise 10 or moreamino acids based on or derived from a polypeptide sequence selectedfrom a group comprising SEQ ID NO: 15-19. The one or more TAs maycomprise an amino acid sequence that is at least about 50% homologous toan amino acid sequence selected from the group comprising SEQ ID NO:15-19. The one or more TAs may comprise an amino acid sequence that isat least 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 15-19. The one or more TAs may comprise atleast a portion of a polypeptide sequence selected from a groupcomprising SEQ ID NO: 20-29. The one or more TAs may comprise 10 or moreamino acids based on or derived from a polypeptide sequence selectedfrom a group comprising SEQ ID NO: 20-29. The one or more TAs maycomprise an amino acid sequence that is at least about 50% homologous toan amino acid sequence selected from the group comprising SEQ ID NO:20-29. The one or more TAs may comprise an amino acid sequence that isat least 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 20-29. The one or more TAs may comprise atleast a portion of a polypeptide sequence selected from a groupcomprising SEQ ID NO: 30-53. The one or more TAs may comprise 10 or moreamino acids based on or derived from a polypeptide sequence selectedfrom a group comprising SEQ ID NO: 30-53. The one or more TAs maycomprise an amino acid sequence that is at least about 50% homologous toan amino acid sequence selected from the group comprising SEQ ID NO:30-53. The one or more TAs may comprise an amino acid sequence that isat least 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 30-53.

Further disclosed herein are peptide lipid conjugates having thestructure of Formula (II):PC-A¹-P¹-L   Formula (II)wherein

PC is a peptide conjugate;

A¹ is a chemical group linking PC and P¹;

P¹ is a bond or -PEG-A²-;

PEG is a chemical group comprising one or more polyethylene glycolsubunits;

A² is a chemical group linking PEG and L; and

L is a lipid.

The PC of Formula (II) may comprise a peptide region comprising one ormore peptide therapeutic agents (TAs) and one or more staples. The oneor more staples may connect to two or more residues in the peptideregion. The staple may connect two or more residues on the same TA. Thestaple may connect two or more residues on two or more TAs. Two or morestaples may connect two or more residues on the same TA. Two or morestaples may connect two or more residues on two or more TAs. The two ormore residues may comprise cysteine.

Further disclosed herein are peptide lipid conjugates having thestructure of Formula (III):

wherein:

-   -   TA is a therapeutic agent;    -   each Q is the same or different, and is a staple;    -   each A¹ is the same or different, and is a chemical group        linking Q and P¹;    -   each P¹ is a bond or -PEG-A²-;    -   each PEG is the same or different, and is a chemical group        comprising one or more polyethylene glycol subunits;    -   each A² is the same or different, and is a chemical group        linking PEG and L;    -   each L is the same or different, and is a lipid;    -   a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   b is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The PLC of Formula (III) may comprise one or more staples. The one ormore staples may connect to two or more residues in the peptide region.The staple may connect two or more residues on the same TA. The two ormore residues may comprise cysteine.

Further disclosed herein are peptide lipid conjugates having thestructure of Formula (IV):

wherein:

-   -   TA is a therapeutic agent;    -   each Q is the same or different, and is a staple;    -   each A¹ is the same or different, and is a chemical group        linking Q and P¹;    -   each P¹ is a bond or -PEG-A²-;    -   each PEG is the same or different, and is a chemical group        comprising one or more polyethylene glycol subunits;    -   each A² is the same or different, and is a chemical group        linking PEG and L;    -   each L is the same or different, and is a lipid;    -   a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   b is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   c is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The PLC of Formula (IV) may comprise one or more staples. The one ormore staples may connect to two or more residues in the peptide region.The staple may connect two or more residues on the same TA. The two ormore residues may comprise cysteine.

The P¹ of the PLC of Formula (II), Formula (III), or Formula (IV) maycomprise PEG. PEG may be selected from

wherein m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20.

The PLC of Formula (II), Formula (III), or Formula (IV) may comprise anA¹ selected from

Each R¹, R², R³, and R⁴ may be independently selected from H, halo, CN,—SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵. Each R⁵ may beindependently selected from H, alkyl, haloalkyl, arylalkyl, andheteroalkyl. K may be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; p may be 2, 3, 4,5, 6, 7, 8, 9, or 10; and q may be 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The PLC of Formula (II), Formula (III), or Formula (IV) may comprise anA² selected from a bond,

X may be a bond, NR⁵, S, or O. R¹, R², R³, and R⁴ may be independentlyselected from H, halo, CN, —SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵,and —OR⁵. R⁵ may be H, alkyl, haloalkyl, arylalkyl, or heteroalkyl. R⁶may be H, alkyl, arylalkyl, —(CR¹R²)_(t)SR⁵, —(CR¹R²)_(t)NR⁵R⁵,—(CR¹R²)_(t)OR⁵, or —(CR¹R²)_(t)CO₂R⁵. Each R⁷ may be independentlyselected from H, alkyl, haloalkyl, arylalkyl, and heteroalkyl. R may be1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. S may be 1, 2, 3, 4, or 5. T may be 0,1, 2, 3, 4, or 5.

The PLC of Formula (II), Formula (III), or Formula (IV) may comprise aP¹ comprising -PEG-A².

The PLCs disclosed herein may comprise one or more staples prepared fromone or more precursor compounds comprising one or more:

wherein

-   -   each W is independently selected from Cl, Br, I, and maleimide;    -   each X is independently selected from O, NH, and S;    -   each Y is independently selected from N and CH;    -   each Z is independently selected from N and CH;    -   a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or        17;    -   b is 1, 2, 3, 4, 5, or 6; and    -   c is 1 or 2.

The A¹ of the PLC of Formula (II) may be connected to PC via a chemicalbond between A¹ and a functional group of a residue of PC. The A¹ of thePLC of Formula (II) may be connected to PC via a chemical bond betweenA¹ and the sulfur atom of a cysteine residue of PC. The A¹ of the PLC ofFormula (II) may be connected to PC at the staple.

Each of the one or more lipids of the PLC of Formula (II) may beattached to the one or more staples to form a lipid staple precursorprior to forming the peptide conjugate. The lipid staple precursor maybe

wherein each W is independently selected from Cl, Br, I, and maleimide;c is 1 or 2; and L is the lipid.

Further disclosed herein are methods of producing a peptide lipidconjugate (PLC) of Formula (II): PC-A¹-P¹-L, wherein PC is a peptideconjugate comprising (a) one or more peptide regions comprising one ormore peptide therapeutic agents (TAs); and (b) one or more staples,wherein the staples connect two or more residues in the peptide region;A¹ is a chemical group linking PC and P¹; P¹ is a bond or -PEG-A²-; PEGis a chemical group comprising one or more polyethylene glycol subunits;A² is a chemical group linking PEG and L; and L is a lipid. The two ormore residues in the peptide region may comprise cysteine. The methodmay comprise reacting a cysteine residue of PC or a derivatizablefunctional group of the staple with A³-P¹-L, wherein A³ is a reactiveprecursor to form A¹. A³ may be a haloacetamide, maleimide, benzylhalide, alkyl disulfide, or pyridyl disulfide. A³ may be ahaloacetamide. A³ may be a bromoacetamide.

Further disclosed herein are methods of producing a peptide lipidconjugate (PLC) of Formula (III). The two or more residues in thepeptide region of the PLC of Formula (III) may comprise cysteine. Themethod may comprise reacting a derivatizable functional group of thestaple with A³-P¹-L, wherein A³ is a reactive precursor to form A¹. A³may be a haloacetamide, maleimide, benzyl halide, alkyl disulfide, orpyridyl disulfide. A³ may be a haloacetamide. A³ may be abromoacetamide. A³ may be an alkyl disulfide.

Further disclosed herein are methods of producing a peptide lipidconjugate (PLC) of Formula (IV). The two or more residues in the peptideregion of the PLC of Formula (IV) may comprise cysteine. The method maycomprise reacting a cysteine residue of PC or a derivatizable functionalgroup of the staple with A³-P¹-L, wherein A³ is a reactive precursor toform A¹. The method may comprise reacting a cysteine residue of PC and aderivatizable functional group of the staple with A³-P¹-L, wherein A³ isa reactive precursor to form A¹. A³ may be a haloacetamide, maleimide,benzyl halide, alkyl disulfide, or pyridyl disulfide. A³ may be ahaloacetamide. A³ may be a bromoacetamide. A³ may be an alkyl disulfide.

Disclosed herein are compositions comprising one or more PLCs, whereinthe one or more peptide lipid conjugates (PLCs) comprise (a) one or morelipids; and (b) one or more peptide conjugates (PC), the peptideconjugate (PC) comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, wherein the one or more lipids are attached to theone or more peptide conjugates. The one or more lipids may be attachedto the one or more TAs. The one or more lipids may be attached to theone or more staples. The composition may further comprise one or morepharmaceutically acceptable salts, excipients or vehicles. The two ormore residues in the peptide region may comprise cysteine.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) oneor more lipids, the lipids selected from a group consisting of sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or morepeptide conjugates (PC), the peptide conjugate comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more peptide conjugates. The oneor more lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. The composition mayfurther comprise one or more pharmaceutically acceptable salts,excipients or vehicles. The two or more residues in the peptide regionmay comprise cysteine.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) oneor more lipids; and (b) one or more peptide conjugates (PC), wherein thepeptide conjugate comprises a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, the one or more peptide therapeutic agentscomprising one or more oxyntomodulin, exenatide, exendin-4,glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, a GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof, wherein the one or more lipids areattached to the one or more peptide conjugates. The one or more lipidsmay be attached to the one or more TAs. The one or more lipids may beattached to the one or more staples. The composition may furthercomprise one or more pharmaceutically acceptable salts, excipients orvehicles. The two or more residues in the peptide region may comprisecysteine.

Further disclosed herein are compositions comprising one or more peptidelipid conjugates (PLCs) of Formula (II): PC-A¹-P¹-L, wherein PC is apeptide conjugate comprising (a) one or more peptide regions comprisingone or more peptide therapeutic agents (TAs); and (b) one or morestaples, wherein the staples connect two or more residues in the peptideregion; A¹ is a chemical group linking PC and P¹; P¹ is a bond or-PEG-A²-; PEG is a chemical group comprising one or more polyethyleneglycol subunits; A² is a chemical group linking PEG and L; and L is alipid. The two or more residues in the peptide region may comprisecysteine.

Disclosed herein are compositions comprising one or more PLCs, whereinthe one or more peptide lipid conjugates (PLCs) comprise (a) one or morelipids; and (b) one or more peptide conjugates (PC), the peptideconjugate (PC) comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, wherein the one or more lipids are attached to theone or more staples. The composition may further comprise one or morepharmaceutically acceptable salts, excipients or vehicles. At least oneof the two or more residues may be cysteine. The two or more residues inthe peptide region may comprise cysteine. The two or more residues maybe at least about 4 amino acid residues apart. The two or more residuesmay be at least about 7 amino acid residues apart. The two or moreresidues may be at least about 11 amino acid residues apart. The one ormore lipids may be attached to the one or more therapeutic agents. Theone or more lipids may be attached to the one or more staples.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) oneor more lipids, the lipids selected from a group consisting of sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or morepeptide conjugates (PC), the peptide conjugate comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more staples. The composition mayfurther comprise one or more pharmaceutically acceptable salts,excipients or vehicles. At least one of the two or more residues may becysteine. The two or more residues in the peptide region may comprisecysteine. The two or more residues may be at least about 4 amino acidresidues apart. The two or more residues may be at least about 7 aminoacid residues apart. The two or more residues may be at least about 11amino acid residues apart. The one or more lipids may be attached to theone or more therapeutic agents. The one or more lipids may be attachedto the one or more staples.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) oneor more lipids; and (b) one or more peptide conjugates (PC), wherein thepeptide conjugate comprises a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, the one or more peptide therapeutic agentscomprising one or more oxyntomodulin, exenatide (exendin-4),glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, a GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof, wherein the one or more lipids areattached to the one or more staples. The composition may furthercomprise one or more pharmaceutically acceptable salts, excipients orvehicles. At least one of the two or more residues may be cysteine. Thetwo or more residues in the peptide region may comprise cysteine. Thetwo or more residues may be at least about 4 amino acid residues apart.The two or more residues may be at least about 7 amino acid residuesapart. The two or more residues may be at least about 11 amino acidresidues apart. The one or more lipids may be attached to the one ormore therapeutic agents. The one or more lipids may be attached to theone or more staples.

Further disclosed herein are compositions comprising one or more peptidelipid conjugates (PLCs) of Formula (III). The PLC of Formula (III) maycomprise a peptide region comprising a plurality of amino acid residues.At least one of the plurality of amino acid residues may be cysteine.The PLC of Formula (III) may comprise one or more staples. The one ormore staples may connect two or more residues in the peptide region. Thetwo or more residues in the peptide region may comprise cysteine. Thetwo or more residues may be at least about 4 amino acid residues apart.The two or more residues may be at least about 7 amino acid residuesapart. The two or more residues may be at least about 11 amino acidresidues apart. The PLC of Formula (III) may comprise one or morelipids. The PLC of Formula (III) may comprise a peptide regioncomprising one or more therapeutic agents. The one or more lipids may beattached to the one or more therapeutic agents. The one or more lipidsmay be attached to the one or more staples.

Further disclosed herein are compositions comprising one or more peptidelipid conjugates (PLCs) of Formula (IV). The PLC of Formula (IV) maycomprise a peptide region comprising a plurality of amino acid residues.At least one of the plurality of amino acid residues may be cysteine.The PLC of Formula (IV) may comprise one or more staples. The one ormore staples may connect two or more residues in the peptide region. Thetwo or more residues in the peptide region may comprise cysteine. Thetwo or more residues may be at least about 4 amino acid residues apart.The two or more residues may be at least about 7 amino acid residuesapart. The two or more residues may be at least about 11 amino acidresidues apart. The PLC of Formula (IV) may comprise one or more lipids.The PLC of Formula (IV) may comprise a peptide region comprising one ormore therapeutic agents. The one or more lipids may be attached to theone or more therapeutic agents. The one or more lipids may be attachedto the one or more staples.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) twoor more lipids; and (b) one or more peptide conjugates (PC), the peptideconjugate (PC) comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, wherein at least one of the two or more lipids areattached to the one or more therapeutic agents and at least one of thetwo or more lipids are attached to the one or more staples. Thecomposition may further comprise one or more pharmaceutically acceptablesalts, excipients or vehicles. At least one of the two or more residuesmay be cysteine. The two or more residues in the peptide region maycomprise cysteine. The two or more residues may be at least about 4amino acid residues apart. The two or more residues may be at leastabout 7 amino acid residues apart. The two or more residues may be atleast about 11 amino acid residues apart. At least one of the lipids maybe attached to the one or more therapeutic agents. At least one of thelipids may be attached to the one or more staples.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) twoor more lipids, the lipids selected from a group consisting of sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or morepeptide conjugates (PC), the peptide conjugate comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein at least oneof the two or more lipids are attached to the one or more therapeuticagents and at least one of the two or more lipids are attached to theone or more staples. The composition may further comprise one or morepharmaceutically acceptable salts, excipients or vehicles. At least oneof the two or more residues may be cysteine. The two or more residues inthe peptide region may comprise cysteine. The two or more residues maybe at least about 4 amino acid residues apart. The two or more residuesmay be at least about 7 amino acid residues apart. The two or moreresidues may be at least about 11 amino acid residues apart. At leastone of the lipids may be attached to the one or more therapeutic agents.At least one of the lipids may be attached to the one or more staples.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) twoor more lipids; and (b) one or more peptide conjugates (PC), wherein thepeptide conjugate comprises a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, the one or more peptide therapeutic agentscomprising one or more oxyntomodulin, exenatide (exendin-4),glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, a GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof, wherein at least one of the two ormore lipids are attached to the one or more therapeutic agents and atleast one of the two or more lipids are attached to the one or morestaples. The composition may further comprise one or morepharmaceutically acceptable salts, excipients or vehicles. At least oneof the two or more residues may be cysteine. The two or more residues inthe peptide region may comprise cysteine. The two or more residues maybe at least about 4 amino acid residues apart. The two or more residuesmay be at least about 7 amino acid residues apart. The two or moreresidues may be at least about 11 amino acid residues apart. At leastone of the lipids may be attached to the one or more therapeutic agents.At least one of the lipids may be attached to the one or more staples.

Disclosed herein are methods for treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids; and (b) one or morepeptide conjugates (PC), the peptide conjugate (PC) comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more peptide conjugates. The oneor more lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. At least one of theresidues in the peptide region may be a cysteine. The two or moreresidues in the peptide region may comprise cysteine. The two or moreresidues may be at least about 4 amino acid residues apart. The two ormore residues may be at least about 7 amino acid residues apart. The twoor more residues may be at least about 11 amino acid residues apart.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids, the lipids selectedfrom a group consisting of sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols; and (b) one or more peptide conjugates (PC), the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, wherein the one or more lipids are attached to the oneor more peptide conjugates. The one or more lipids may be attached tothe one or more TAs. The one or more lipids may be attached to the oneor more staples. At least one of the residues in the peptide region maybe a cysteine. The two or more residues in the peptide region maycomprise cysteine. The two or more residues may be at least about 4amino acid residues apart. The two or more residues may be at leastabout 7 amino acid residues apart. The two or more residues may be atleast about 11 amino acid residues apart.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids; and (b) one or morepeptide conjugates (PC), wherein the peptide conjugate comprises apeptide region comprising one or more peptide therapeutic agents (TA)and a staple region comprising one or more staples, the one or morestaples connect two or more residues in the peptide region, the one ormore peptide therapeutic agents comprising one or more oxyntomodulin,exenatide (exendin-4), glucagon-like protein-1 (GLP-1), GLP-2, glucagon,a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dual agonist, a GLP1R,GCGR and GIPR tri-agonis, or a derivative thereof, wherein the one ormore lipids are attached to the one or more peptide conjugates. The oneor more lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. At least one of theresidues in the peptide region may be a cysteine. The two or moreresidues in the peptide region may comprise cysteine. The two or moreresidues may be at least about 4 amino acid residues apart. The two ormore residues may be at least about 7 amino acid residues apart. The twoor more residues may be at least about 11 amino acid residues apart.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more peptide lipid conjugates (PLCs) of Formula (II):PC-A¹-P¹-L, wherein PC is a peptide conjugate comprising (a) one or morepeptide regions comprising one or more peptide therapeutic agents (TAs);and (b) one or more staples, wherein the staples connect two or moreresidues in the peptide region; A¹ is a chemical group linking PC andP¹; P¹ is a bond or -PEG-A²-; PEG is a chemical group comprising one ormore polyethylene glycol subunits; A² is a chemical group linking PEGand L; and L is a lipid. L may be attached to the one or more TAs. L maybe attached to the one or more staples. At least one of the residues inthe peptide region may be a cysteine. The two or more residues in thepeptide region may comprise cysteine. The two or more residues may be atleast about 4 amino acid residues apart. The two or more residues may beat least about 7 amino acid residues apart. The two or more residues maybe at least about 11 amino acid residues apart.

Disclosed herein are methods for treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids; and (b) one or morepeptide conjugates (PC), the peptide conjugate (PC) comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more staples. The one or morelipids may be attached to the one or more TAs. The one or more lipidsmay be attached to the one or more staples. At least one of the residuesin the peptide region may be a cysteine. The two or more residues in thepeptide region may comprise cysteine. The two or more residues may be atleast about 4 amino acid residues apart. The two or more residues may beat least about 7 amino acid residues apart. The two or more residues maybe at least about 11 amino acid residues apart.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids, the lipids selectedfrom a group consisting of sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols; and (b) one or more peptide conjugates (PC), the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, wherein the one or more lipids are attached to the oneor more staples. The one or more lipids may be attached to the one ormore TAs. The one or more lipids may be attached to the one or morestaples. At least one of the residues in the peptide region may be acysteine. The two or more residues in the peptide region may comprisecysteine. The two or more residues may be at least about 4 amino acidresidues apart. The two or more residues may be at least about 7 aminoacid residues apart. The two or more residues may be at least about 11amino acid residues apart.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids; and (b) one or morepeptide conjugates (PC), wherein the peptide conjugate comprises apeptide region comprising one or more peptide therapeutic agents (TA)and a staple region comprising one or more staples, the one or morestaples connect two or more residues in the peptide region, the one ormore peptide therapeutic agents comprising one or more oxyntomodulin,exenatide, exendin-4, glucagon-like protein-1 (GLP-1), GLP-2, glucagon,a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dual agonist, a GLP1R,GCGR and GIPR tri-agonist, or a derivative thereof, wherein the one ormore lipids are attached to the one or more staples. The one or morelipids may be attached to the one or more TAs. The one or more lipidsmay be attached to the one or more staples. At least one of the residuesin the peptide region may be a cysteine. The two or more residues in thepeptide region may comprise cysteine. The two or more residues may be atleast about 4 amino acid residues apart. The two or more residues may beat least about 7 amino acid residues apart. The two or more residues maybe at least about 11 amino acid residues apart.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more peptide lipid conjugates (PLCs) of Formula (III).The PLC of Formula (III) may comprise one or more lipids. The PLC ofFormula (III) may comprise one or more therapeutic agents (TAs). The PLCof Formula (III) may comprise one or more staples. The one or morelipids may be attached to the one or more TAs. The one or more lipidsmay be attached to the one or more staples. At least one of the residuesin the peptide region may be a cysteine. The two or more residues in thepeptide region may comprise cysteine. The two or more residues may be atleast about 4 amino acid residues apart. The two or more residues may beat least about 7 amino acid residues apart. The two or more residues maybe at least about 11 amino acid residues apart.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more peptide lipid conjugates (PLCs) of Formula (IV). ThePLC of Formula (IV) may comprise one or more lipids. The PLC of Formula(IV) may comprise one or more peptide regions. The one or more peptideregions may comprise one or more therapeutic agents (TAs). The PLC ofFormula (IV) may comprise one or more staples. The one or more lipidsmay be attached to the one or more TAs. The one or more lipids may beattached to the one or more staples. The one or more staples may connecttwo or more residues in the peptide region. At least one of the residuesin the peptide region may be a cysteine. The two or more residues in thepeptide region may comprise cysteine. The two or more residues may be atleast about 4 amino acid residues apart. The two or more residues may beat least about 7 amino acid residues apart. The two or more residues maybe at least about 11 amino acid residues apart.

Disclosed herein are methods for treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) two or more lipids; and (b) one or morepeptide conjugates (PC), the peptide conjugate (PC) comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein at least oneof the two or more lipids are attached to the one or more therapeuticagents and at least one of the two or more lipids are attached to theone or more staples. The one or more lipids may be attached to the oneor more TAs. The one or more lipids may be attached to the one or morestaples. At least one of the residues in the peptide region may be acysteine. The two or more residues in the peptide region may comprisecysteine. The two or more residues may be at least about 4 amino acidresidues apart. The two or more residues may be at least about 7 aminoacid residues apart. The two or more residues may be at least about 11amino acid residues apart.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) two or more lipids, the lipids selectedfrom a group consisting of sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols; and (b) one or more peptide conjugates (PC), the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, wherein at least one of the two or more lipids areattached to the one or more therapeutic agents and at least one of thetwo or more lipids are attached to the one or more staples. The one ormore lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. At least one of theresidues in the peptide region may be a cysteine. The two or moreresidues in the peptide region may comprise cysteine. The two or moreresidues may be at least about 4 amino acid residues apart. The two ormore residues may be at least about 7 amino acid residues apart. The twoor more residues may be at least about 11 amino acid residues apart.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) two or more lipids; and (b) one or morepeptide conjugates (PC), wherein the peptide conjugate comprises apeptide region comprising one or more peptide therapeutic agents (TA)and a staple region comprising one or more staples, the one or morestaples connect two or more residues in the peptide region, the one ormore peptide therapeutic agents comprising one or more oxyntomodulin,exenatide, exendin-4, glucagon-like protein-1 (GLP-1), GLP-2, glucagon,a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dual agonist, a GLP1R,GCGR and GIPR tri-agonist, or a derivative thereof, wherein at least oneof the two or more lipids are attached to the one or more therapeuticagents and at least one of the two or more lipids are attached to theone or more staples. The one or more lipids may be attached to the oneor more TAs. The one or more lipids may be attached to the one or morestaples. At least one of the residues in the peptide region may be acysteine. The two or more residues in the peptide region may comprisecysteine. The two or more residues may be at least about 4 amino acidresidues apart. The two or more residues may be at least about 7 aminoacid residues apart. The two or more residues may be at least about 11amino acid residues apart.

The PLCs disclosed herein may be used to treat a disease or condition.The disease or condition may be diabetes or obesity, or a medicalcondition associated with diabetes or obesity. The disease or conditionmay be non-alcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), or cardiovascular disease. The disease orcondition may be short bowel syndrome (SBS), inflammatory bowel disease(IBD), psoriasis, ulcerative colitis, or Crohn's disease. The disease orcondition may be Alzheimer's disease, Parkinson's disease orHuntington's disease. The PLCs disclosed herein may be administered withone or more additional therapeutic agents. The one or more additionaltherapeutic agents may comprise one or more anti-inflammatory drugs,statins, diuretics, beta-blockers, angiotensin converting enzymeinhibitors, or angiotensin II receptor blockers. The one or moreadditional therapeutic agents may be aspirin. The one or more additionaltherapeutic agents may be selected from a group consisting of otherdiabetes drugs, DPP4 inhibitors, SGLT2 inhibitors, hypoglycemic drugsand biguanidine drugs, insulin secretogogues and sulfonyl urea drugs,TZD drugs, insulin and insulin analogs, FGF21 and analogs, leptin orleptin analogs, amylin and amylin analogs, an anti-inflammatory drug,cyclosporine A or FK506, 5-ASA, and a statin, or any combinationthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. The invention is best understood from the followingdetailed description when read in conjunction with the accompanyingdrawings. It is emphasized that, according to common practice, thevarious features of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1A depicts an exemplary peptide conjugate comprising onetherapeutic agent (TA) and one staple (C).

FIG. 1B depicts an exemplary peptide conjugate comprising twotherapeutic agents (TA¹ and TA²) and one staple (C).

FIG. 1C depicts an exemplary peptide conjugate comprising twotherapeutic agents (TA¹ and TA²) and one staple containing two moieties(C¹ and C²).

FIG. 1D depicts an exemplary peptide conjugate comprising twotherapeutic agents (TA¹ and TA²) and two staples (C¹ and C²).

FIG. 1E depicts an exemplary peptide conjugate comprising onetherapeutic agent (TA) and one staple containing two moieties (C¹ andC²).

FIG. 1F depicts an exemplary peptide conjugate comprising onetherapeutic agent (TA) and one staple (C).

FIG. 2A depicts an exemplary peptide lipid conjugate comprising onepeptide conjugate (PC) and one half-life extending molecule comprising alipid (L).

FIG. 2B depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and one half-life extendingmolecule comprising a lipid (L).

FIG. 2C depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and one half-life extendingmolecule comprising a lipid (L).

FIG. 2D depicts an exemplary peptide lipid conjugate comprising twotherapeutic agents (TA¹ and TA²), one staple (C), and one half-lifeextending molecule comprising a lipid (L).

FIG. 2E depicts an exemplary peptide lipid conjugate comprising twotherapeutic agents (TA¹ and TA²), one staple (C¹), and one half-lifeextending molecule comprising a lipid (L).

FIG. 2F depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and one half-life extendingmolecule comprising a lipid linked to the therapeutic agent.

FIG. 2G depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and one half-life extendingmolecule comprising a lipid (L) linked to the staple (C).

FIG. 3A depicts an exemplary staple-lipid construct comprising a staple(C) and one half-life extending molecule comprising a lipid (L).

FIG. 3B depicts an exemplary staple-lipid construct comprising a staple(C) and one half-life extending molecule comprising a lipid (L).

FIG. 3C depicts an exemplary staple-lipid construct comprising a staple(C) and one half-life extending molecule comprising a lipid (L).

FIG. 3D depicts an exemplary staple-lipid construct comprising a staple(C) and two half-life extending molecules comprising a lipid (L¹ andL²).

FIG. 3E depicts an exemplary staple-lipid construct comprising a staple(C) and two half-life extending molecules comprising a lipid (L¹ andL²).

FIG. 3F depicts an exemplary staple-lipid construct comprising a staple(C) and two half-life extending molecules comprising a lipid (L¹ andL²).

FIG. 3G depicts an exemplary staple-lipid construct comprising a staple(C) and three half-life extending molecules comprising a lipid (L¹, L²,and L³).

FIG. 3G depicts an exemplary staple-lipid construct comprising a staple(C) and four half-life extending molecules comprising a lipid (L¹, L²,L³, and L⁴).

FIG. 4A depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and one half-life extendingmolecule comprising a lipid (L) linked to the therapeutic agent.

FIG. 4B depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and one half-life extendingmolecule comprising a lipid (L) linked to the therapeutic agent.

FIG. 4C depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and one half-life extendingmolecule comprising a lipid (L) linked to the therapeutic agent.

FIG. 4D depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and two half-life extendingmolecules comprising a lipid (L¹ and L²) linked to the therapeuticagent.

FIG. 4E depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and two half-life extendingmolecules comprising a lipid (L¹ and L²) linked to the therapeuticagent.

FIG. 4F depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and two half-life extendingmolecules comprising a lipid (L¹ and L²) linked to the therapeuticagent.

FIG. 4G depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and three half-life extendingmolecules comprising a lipid (L¹, L², and L³) linked to the therapeuticagent.

FIG. 4H depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and four half-life extendingmolecules comprising a lipid (L¹, L², L³, and L⁴) linked to thetherapeutic agent.

FIG. 5A depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and one half-life extendingmolecule comprising a lipid (L) linked to the staple.

FIG. 5B depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and one half-life extendingmolecule comprising a lipid (L) linked to the staple.

FIG. 5C depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and one half-life extendingmolecule comprising a lipid (L) linked to the staple.

FIG. 5D depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and two half-life extendingmolecules comprising a lipid (L¹ and L²) linked to the staple.

FIG. 5E depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and two half-life extendingmolecules comprising a lipid (L¹ and L²) linked to the staple.

FIG. 5F depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and two half-life extendingmolecules comprising a lipid (L¹ and L²) linked to the staple.

FIG. 5G depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and three half-life extendingmolecules comprising a lipid (L¹, L², and L³) linked to the staple.

FIG. 5H depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), and four half-life extendingmolecules comprising a lipid (L¹, L², L³, and L⁴) linked to the staple.

FIG. 6A depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), one half-life extending moleculecomprising a lipid (L¹) linked to the staple, and one half-lifeextending molecule comprising a lipid (L²) linked to the therapeuticagent.

FIG. 6B depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), one half-life extending moleculecomprising a lipid (L¹) linked to the staple, and one half-lifeextending molecule comprising a lipid (L²) linked to the therapeuticagent.

FIG. 6C depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), one half-life extending moleculecomprising a lipid (L¹) linked to the staple, and one half-lifeextending molecule comprising a lipid (L²) linked to the therapeuticagent.

FIG. 6D depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), one half-life extending moleculecomprising a lipid (L¹) linked to the staple, and two half-lifeextending molecules comprising a lipid (L² and L³) linked to thetherapeutic agent.

FIG. 6E depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), two half-life extendingmolecules comprising a lipid (L¹ and L²) linked to the staple, and twohalf-life extending molecules comprising a lipid (L³ and L⁴) linked tothe therapeutic agent.

FIG. 6F depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), two half-life extendingmolecules comprising a lipid (L¹ and L²) linked to the staple, and twohalf-life extending molecules comprising a lipid (L³ and L⁴) linked tothe therapeutic agent.

FIG. 6G depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), one half-life extending moleculecomprising a lipid (L¹) linked to the staple, and three half-lifeextending molecules comprising a lipid (L², L³, and L⁴) linked to thetherapeutic agent.

FIG. 6H depicts an exemplary peptide lipid conjugate comprising onetherapeutic agent (TA), one staple (C), two half-life extendingmolecules comprising a lipid (L¹ and L²) linked to the staple, and fourhalf-life extending molecules comprising a lipid (L³, L⁴, L⁵, and L⁶)linked to the therapeutic agent.

FIG. 7 depicts thioether-based macrolactonization via two differentprotocols.

FIG. 8 shows dosage response curves for GLP-1R CRE luciferase assay.

FIG. 9 shows dosage response curves for GCGR CRE luciferase assay.

FIG. 10 shows dosage response curves for GLP-2R CRE luciferase assay.

FIG. 11 shows dosage response curves for GIPR CRE luciferase assay.

FIG. 12A shows the pharmacokinetic profiles of mTA4 in mice after i.v.or s.c. injection.

FIG. 12B shows the pharmacokinetic profile of mTA37 in mice after i.v.or s.c. injection.

FIG. 12C shows the pharmacokinetic profile of mTA68 in mice after i.v.or s.c. injection.

FIG. 13A shows the oral glucose tolerance test (OGTT) results for miceafter mTA4 administration.

FIG. 13B shows the AUC from the oral glucose tolerance test (OGTT)results in mice after mTA4 administration.

FIG. 13C shows the oral glucose tolerance test (OGTT) results for miceafter mTA37 administration.

FIG. 13D shows the AUC from the oral glucose tolerance test (OGTT)results in mice after mTA37 administration.

FIG. 14A shows the dose response weight loss in DIO mice after mTA4administration.

FIG. 14B shows the dose response weight loss in DIO mice after mTA37 andmTA4 administration.

FIG. 15A shows the body weights in DIO animals with daily s.c. dosingwith mTA4 and mTA37.

FIG. 15B shows the cumulative food intake in DIO animals with daily s.c.dosing with mTA4 and mTA37.

FIG. 16A shows the intraperitoneal glucose tolerance test (IPGTT)results in DIO mice after mTA4 and mTA37 administration.

FIG. 16B shows the AUC from the intraperitoneal glucose tolerance test(IPGTT) results for mice after mTA4 and mTA37 administration.

FIG. 17 shows fasting blood glucose levels after mTA administration.

FIG. 18A shows the plasma triglyceride levels after mTA4 and mTA37administration.

FIG. 18B shows the plasma cholesterol levels after mTA4 and mTA37administration.

FIG. 19 shows lipid droplet staining in liver after mTA administration.

FIG. 20 shows pharmacokinetic profiles of mTA4 after i.v. or s.c.injection, and micro-needle based transdermal delivery in guinea pigs.

FIG. 21A shows the OGTT results after mTA4 administration throughmicro-needle based transdermal delivery in guinea pigs (24 h postinjection).

FIG. 21B shows OGTT results after mTA4 administration throughmicro-needle based transdermal delivery in guinea pigs (48 h postinjection).

FIG. 21C shows OGTT results after mTA4 administration throughmicro-needle based transdermal delivery in guinea pigs (96 h postinjection).

FIG. 22A shows the AUC values from OGTT results after mTA4administration through micro-needle based transdermal delivery in guineapigs (24 h post injection).

FIG. 22B shows AUC values from OGTT results after mTA4 administrationthrough micro-needle based transdermal delivery in guinea pigs (48 hpost injection).

FIG. 22C shows AUC values from OGTT results after mTA4 administrationthrough micro-needle based transdermal delivery in guinea pigs (96 hpost injection).

FIG. 23A shows the body weight changes in DSS-induced colitis mice afterdaily administration of mTA68 for 12 days.

FIG. 23B shows the intestine weight vs. body weight in DSS-inducedcolitis mice after daily administration of mTA68 for 12 days.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein, in some embodiments, are compounds comprising: atherapeutic peptide, a staple and a non-peptide molecule, wherein thestaple is conjugated to one or more amino acids of the therapeuticpeptide. A first region of a staple may be conjugated to a first aminoacid of the therapeutic peptide and a second region of a staple may beconjugated to a second amino acid of a therapeutic peptide. Thenon-peptide molecule may not be conjugated to the therapeutic peptide.The non-peptide molecule may be conjugated to the staple. Thenon-peptide molecule may be conjugated to the staple such that thenon-peptide molecule is distal to an active site or binding site of thetherapeutic peptide. The staple may stabilize the therapeutic peptidewhile providing a conjugation site for the non-peptide molecule, suchthat the non-peptide molecule does not hinder and/or interfere with thetherapeutic peptide binding to a target. A property of the therapeuticpeptide in the composition may be different than a respective propertyof the therapeutic peptide alone. A property of the therapeutic peptidein the composition may be different than a respective property of thetherapeutic peptide that is conjugated to the non-peptide molecule. Theproperty may be selected from an absorption rate constant, an absorptionefficiency, an elimination rate constant, a half-life, a bindingaffinity, a binding efficiency, a disassociation constant, a targetselectivity and a potency and in vivo efficacy. A therapeutic effect ofthe compound may be greater than a therapeutic effect of the therapeuticpeptide alone. A therapeutic effect of the compound may be greater thana therapeutic effect of a respective therapeutic peptide that isconjugated to the non-peptide molecule. A therapeutic effect of thecompound may be longer-lasting than a therapeutic effect of thetherapeutic peptide alone. A therapeutic effect of the composition maybe longer-lasting than a therapeutic effect of a respective therapeuticpeptide that is conjugated to the non-peptide molecule. The staple maystabilize the therapeutic peptide. The non-peptide molecule may extend ahalf-life of the therapeutic peptide. The non-peptide molecule maycomprise a lipid moiety. The non-peptide molecule may comprise apolyethylene glycol unit.

Disclosed herein are modified therapeutic agents (mTAs). Generally, themTA may comprise a therapeutic agent (TA), a staple, and a half-lifeextending molecule (HEM). The TA may be a modified or unmodifiedtherapeutic peptide. The TA may be a modified therapeutic peptide. Themodified therapeutic peptide may be a derivative of a peptide selectedfrom GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP; thederivative being a peptide comprising one or more amino acid additions,deletions, or substitutions, or a combination thereof. The TA may be anunmodified therapeutic peptide. The unmodified therapeutic peptide maybe selected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, andGIP. The staple may be covalently attached to the TA. The HEM may beattached to the staple. The HEM may be attached to the TA. The staplemay be attached to the TA via two amino acid residues on the modified orunmodified therapeutic peptide. One or both of the two amino acidresidues may be an amino acid addition or substitution on the modifiedtherapeutic peptide. One or both of the two amino acid residues may becysteine. The two or more amino acid residues may be at least about 4amino acid residues apart. The two or more amino acid residues may be atleast about 7 amino acid residues apart. The two or more amino acidresidues may be at least about 11 amino acid residues apart. The HEM maycomprise a lipid, a polyglycol region, or a combination of both. The HEMmay comprise a lipid. The HEM may comprise a lipid and a polyglycolregion. The HEM may comprise a polyglycol region. The HEM may comprise apeptide or protein. The lipid may be selected from a group consisting ofsterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, fatty amines, and fatty alcohols,and derivatives thereof. The polyglycol region may comprise one or morepolyethylene glycol units, polypropylene glycol units, or polybutyleneglycol units, or a combination thereof. The mTA may have more than onestaple. The mTA may have more than one HEM. The mTA may have a longerhalf-life than the half-life of the unmodified therapeutic peptidealone. The mTA may have a higher potency than the potency of theunmodified therapeutic peptide alone. The mTA may have a higher targetselectivity than the target selectivity of the unmodified therapeuticpeptide alone. The mTA may have a higher binding affinity than thebinding affinity of the unmodified therapeutic peptide alone.

Disclosed herein are peptide lipid conjugates (PLCs) which comprise oneor more therapeutic agents (TAs), one or more half-life extendingmolecules (HEMs), and one or more staples that are directly attached tothe one or more TAs. The one or more HEMs may comprise one or morelipids. The one or more HEMS may further comprise one or morepolyethylene glycol subunits, wherein the one or more lipids areattached to the one or more polyethylene glycol subunits. Alternatively,the one or more HEMS may comprise one or more polyethylene glycolsubunits. The one or more HEMS may comprise one or more peptides orproteins. The one or more HEMs may comprise one or more moleculesselected from lipids, polyethylene glycol subunits, peptides, orproteins, or any combination thereof. The one or more HEMs may beattached directly to the TA. The one or more HEMs may be attacheddirectly to the staple. The HEMS may be attached directly to the TA andto the staple. The HEM may be a lipid. The PLCs may comprise two or moretherapeutic agents. The PLCs may comprise two or more staples. The PLCsmay comprise two or more HEMs. The PLCs may comprise a plurality of HEMsattached to the one or more therapeutic agents. The PLCs may comprise aplurality of HEMs attached to the one or more staples.

Generally, the PLCs comprise one or more lipids attached to one or morepeptide conjugates (PCs). The PLCs may comprise (a) one or more lipids;and (b) one or more peptide conjugates (PC), the peptide conjugate (PC)comprising a peptide region comprising one or more peptide therapeuticagents (TA) and a staple region comprising one or more staples, the oneor more staples connect two or more residues in the peptide region,wherein the one or more lipids are attached to the one or more peptideconjugates. The two or more residues in the peptide region may comprisecysteine. The one or more lipids may be attached to the one or morestaples in the peptide region. Alternatively or additionally, the one ormore lipids may be attached to the one or more peptide therapeuticagents in the peptide region.

The peptide lipid conjugates (PLCs) may comprise (a) one or more lipids,the lipids selected from a group consisting of sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, and fatty alcohols; and (b) one or more peptideconjugates (PC), the peptide conjugate comprising a peptide regioncomprising one or more peptide therapeutic agents (TA) and a stapleregion comprising one or more staples, the one or more staples connecttwo or more residues in the peptide region, wherein the one or morelipids are attached to the one or more peptide conjugates. The one ormore lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. At least one of theresidues in the peptide region may be cysteine. The two or more residuesin the peptide region may comprise cysteine. The two or more residuesmay be at least about 4 amino acids apart. The two or more residues maybe at least about 7 amino acids apart. The two or more residues may beat least about 11 amino acids apart.

The PLCs may comprise comprising (a) one or more lipids; and (b) one ormore peptide conjugates (PC), wherein the peptide conjugate comprises apeptide region comprising one or more peptide therapeutic agents (TA)and a staple region comprising one or more staples, the one or morestaples connect two or more residues in the peptide region, the one ormore peptide therapeutic agents comprising one or more oxyntomodulin,exenatide, exendin-4, glucagon-like protein-1 (GLP-1), GLP-2, glucagon,a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dual agonist, or aderivative thereof, wherein the one or more lipids are attached to theone or more peptide conjugates. The one or more lipids may be attachedto the one or more TAs. The one or more lipids may be attached to theone or more staples. At least one of the residues in the peptide regionmay be cysteine. The two or more residues in the peptide region maycomprise cysteine. The two or more residues may be at least about 4amino acids apart. The two or more residues may be at least about 7amino acids apart. The two or more residues may be at least about 11amino acids apart.

Disclosed herein are compositions comprising one or more PLCs, whereinthe one or more peptide lipid conjugates (PLCs) comprise (a) one or morelipids; and (b) one or more peptide conjugates (PC), the peptideconjugate (PC) comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, wherein the one or more lipids are attached to theone or more peptide conjugates. The composition may further comprise oneor more pharmaceutically acceptable salts, excipients or vehicles. Theone or more lipids may be attached to the one or more TAs. The one ormore lipids may be attached to the one or more staples. At least one ofthe residues in the peptide region may be cysteine. The two or moreresidues in the peptide region may comprise cysteine. The two or moreresidues may be at least about 4 amino acids apart. The two or moreresidues may be at least about 7 amino acids apart. The two or moreresidues may be at least about 11 amino acids apart.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) oneor more lipids, the lipids selected from a group consisting of sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or morepeptide conjugates (PC), the peptide conjugate comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more peptide conjugates. Thecomposition may further comprise one or more pharmaceutically acceptablesalts, excipients or vehicles. The one or more lipids may be attached tothe one or more TAs. The one or more lipids may be attached to the oneor more staples. At least one of the residues in the peptide region maybe cysteine. The two or more residues in the peptide region may comprisecysteine. The two or more residues may be at least about 4 amino acidsapart. The two or more residues may be at least about 7 amino acidsapart. The two or more residues may be at least about 11 amino acidsapart.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) oneor more lipids; and (b) one or more peptide conjugates (PC), wherein thepeptide conjugate comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, the one or more peptide therapeutic agentscomprising one or more oxyntomodulin, exenatide, exendin-4,glucagon-like protein-1 (GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, or a derivative thereof,wherein the one or more lipids are attached to the one or more peptideconjugates. The composition may further comprise one or morepharmaceutically acceptable salts, excipients or vehicles. The one ormore lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. At least one of theresidues in the peptide region may be cysteine. The two or more residuesin the peptide region may comprise cysteine. The two or more residuesmay be at least about 4 amino acids apart. The two or more residues maybe at least about 7 amino acids apart. The two or more residues may beat least about 11 amino acids apart.

Further disclosed herein are compositions comprising one or more peptidelipid conjugates (PLCs) of Formula (II): PC-A¹-P¹-L, wherein PC is apeptide conjugate comprising (a) one or more peptide regions comprisingone or more peptide therapeutic agents (TAs); and (b) one or morestaples, wherein the staples connect two or more residues in the peptideregion; A¹ is a chemical group linking PC and P¹; P¹ is a bond or-PEG-A²-; PEG is a chemical group comprising one or more polyethyleneglycol subunits; A² is a chemical group linking PEG and L; and L is alipid. The one or more lipids may be attached to the one or more TAs.The one or more lipids may be attached to the one or more staples. Atleast one of the residues in the peptide region may be cysteine. The twoor more residues in the peptide region may comprise cysteine. The two ormore residues may be at least about 4 amino acids apart. The two or moreresidues may be at least about 7 amino acids apart. The two or moreresidues may be at least about 11 amino acids apart.

Further disclosed herein are compositions comprising one or more peptidelipid conjugates (PLCs) of Formula (III). The PLC of Formula (III) maycomprise a peptide region comprising a plurality of amino acid residues.At least one of the plurality of amino acid residues may be cysteine.The PLC of Formula (III) may comprise one or more staples. The one ormore staples may connect two or more residues in the peptide region. Thetwo or more residues in the peptide region may comprise cysteine. Thetwo or more residues may be at least about 4 amino acid residues apart.The two or more residues may be at least about 7 amino acid residuesapart. The two or more residues may be at least about 11 amino acidresidues apart. The PLC of Formula (III) may comprise one or morelipids. The PLC of Formula (III) may comprise a peptide regioncomprising one or more therapeutic agents. The one or more lipids may beattached to the one or more therapeutic agents. The one or more lipidsmay be attached to the one or more staples.

Further disclosed herein are compositions comprising one or more peptidelipid conjugates (PLCs) of Formula (IV). The PLC of Formula (IV) maycomprise a peptide region comprising a plurality of amino acid residues.At least one of the plurality of amino acid residues may be cysteine.The PLC of Formula (IV) may comprise one or more staples. The one ormore staples may connect two or more residues in the peptide region. Thetwo or more residues in the peptide region may comprise cysteine. Thetwo or more residues may be at least about 4 amino acid residues apart.The two or more residues may be at least about 7 amino acid residuesapart. The two or more residues may be at least about 11 amino acidresidues apart. The PLC of Formula (IV) may comprise one or more lipids.The PLC of Formula (IV) may comprise a peptide region comprising one ormore therapeutic agents. The one or more lipids may be attached to theone or more therapeutic agents. The one or more lipids may be attachedto the one or more staples.

Disclosed herein are methods for treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids; and (b) one or morepeptide conjugates (PC), the peptide conjugate (PC) comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more peptide conjugates. The oneor more lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. At least one of theresidues in the peptide region may be cysteine. The two or more residuesin the peptide region may comprise cysteine. The two or more residuesmay be at least about 4 amino acids apart. The two or more residues maybe at least about 7 amino acids apart. The two or more residues may beat least about 11 amino acids apart.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids, the lipids selectedfrom a group consisting of sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols; and (b) one or more peptide conjugates (PC), the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, wherein the one or more lipids are attached to the oneor more peptide conjugates. The one or more lipids may be attached tothe one or more TAs. The one or more lipids may be attached to the oneor more staples. At least one of the residues in the peptide region maybe cysteine. The two or more residues in the peptide region may comprisecysteine. The two or more residues may be at least about 4 amino acidsapart. The two or more residues may be at least about 7 amino acidsapart. The two or more residues may be at least about 11 amino acidsapart. Further disclosed herein are methods for treating a disease orcondition in a subject in need thereof, the method comprisingadministering to the subject one or more PLCs, wherein the one or morepeptide lipid conjugates (PLCs) comprise (a) one or more lipids; and (b)one or more peptide conjugates (PC), wherein the peptide conjugatecomprising a peptide region comprising one or more peptide therapeuticagents (TA) and a staple region comprising one or more staples, the oneor more staples connect two or more residues in the peptide region, theone or more peptide therapeutic agents comprising one or moreoxyntomodulin, exenatide, exendin-4, glucagon-like protein-1 (GLP-1),GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dualagonist, or a derivative thereof, wherein the one or more lipids areattached to the one or more peptide conjugates. The one or more lipidsmay be attached to the one or more TAs. The one or more lipids may beattached to the one or more staples. At least one of the residues in thepeptide region may be cysteine. The two or more residues in the peptideregion may comprise cysteine. The two or more residues may be at leastabout 4 amino acids apart. The two or more residues may be at leastabout 7 amino acids apart. The two or more residues may be at leastabout 11 amino acids apart.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more peptide lipid conjugates (PLCs) of Formula (II):PC-A¹-P¹-L, wherein PC is a peptide conjugate comprising (a) one or morepeptide regions comprising one or more peptide therapeutic agents (TAs);and (b) one or more staples, wherein the staples connect two or moreresidues in the peptide region; A¹ is a chemical group linking PC andP¹; P¹ is a bond or -PEG-A²-; PEG is a chemical group comprising one ormore polyethylene glycol subunits; A² is a chemical group linking PEGand L; and L is a lipid. L may be attached to the one or more TAs. L maybe attached to the one or more staples. At least one of the two or moreresidues in the peptide region may be cysteine. The two or more residuesin the peptide region may comprise cysteine. The two or more residuesmay be at least about 4 amino acids apart. The two or more residues maybe at least about 7 amino acids apart. The two or more residues may beat least about 11 amino acids apart.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more peptide lipid conjugates (PLCs) of Formula (III).The PLC of Formula (III) may comprise a peptide region comprising aplurality of amino acid residues. At least one of the plurality of aminoacid residues may be cysteine. The PLC of Formula (III) may comprise oneor more staples. The one or more staples may connect two or moreresidues in the peptide region. The two or more residues in the peptideregion may comprise cysteine. The two or more residues may be at leastabout 4 amino acid residues apart. The two or more residues may be atleast about 7 amino acid residues apart. The two or more residues may beat least about 11 amino acid residues apart. The PLC of Formula (III)may comprise one or more lipids. The PLC of Formula (III) may comprise apeptide region comprising one or more therapeutic agents. The one ormore lipids may be attached to the one or more therapeutic agents. Theone or more lipids may be attached to the one or more staples.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more peptide lipid conjugates (PLCs) of Formula (IV). ThePLC of Formula (IV) may comprise a peptide region comprising a pluralityof amino acid residues. At least one of the plurality of amino acidresidues may be cysteine. The PLC of Formula (IV) may comprise one ormore staples. The one or more staples may connect two or more residuesin the peptide region. The two or more residues in the peptide regionmay comprise cysteine. The two or more residues may be at least about 4amino acid residues apart. The two or more residues may be at leastabout 7 amino acid residues apart. The two or more residues may be atleast about 11 amino acid residues apart. The PLC of Formula (IV) maycomprise one or more lipids. The PLC of Formula (IV) may comprise apeptide region comprising one or more therapeutic agents. The one ormore lipids may be attached to the one or more therapeutic agents. Theone or more lipids may be attached to the one or more staples.

Before the present methods, kits and compositions are described ingreater detail, it is to be understood that this invention is notlimited to particular method, kit or composition described, as such may,of course, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to be limiting, since the scope of the present inventionwill be limited only by the appended claims. Examples are put forth soas to provide those of ordinary skill in the art with a completedisclosure and description of how to make and use the present invention,and are not intended to limit the scope of what the inventors regard astheir invention nor are they intended to represent that the experimentsbelow are all or the only experiments performed. Efforts have been madeto ensure accuracy with respect to numbers used (e.g. amounts,temperature, etc.) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof, e.g.polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Methods and compositions are provided for producing PLCs that extend thehalf-life of a therapeutic agent. These methods and compositions findtherapeutic use in a number of diseases, for example, diabetes orobesity may be more effectively treated with a half-life extensionmolecule conjugated to a therapeutic peptide than by the therapeuticpeptide alone. These and other objects, advantages, and features of theinvention will become apparent to those persons skilled in the art uponreading the details of the compositions and methods as more fullydescribed below.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

Modified Therapeutic Agent (mTA)

Disclosed herein are modified therapeutic agents (mTAs) comprising atherapeutic agent (TA), a staple, and a half-life extending molecule(HEM). The TA may be a modified or unmodified therapeutic peptide. TheTA may be covalently attached to the staple. The TA may be covalentlyattached to the staple via two amino acid residues on the modified orunmodified therapeutic peptide. The two amino acid residues may be atleast about 4 amino acid residues apart. The two amino acid residues maybe at least about 7 amino acid residues apart. The two amino acidresidues may be at least about 11 amino acid residues apart. The twoamino acids may be cysteine. The half-life of the mTA may be longer thanthe half-life of the modified or unmodified therapeutic peptide alone.The HEM may comprise a lipid, a polyglycol region, or a combinationthereof. The lipid may be selected from a group consisting of sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, fatty amines, and fatty alcohols, andderivatives thereof. The polyglycol region comprises one or morepolyethylene glycol units, polypropylene glycol units, or polybutyleneglycol units, or a combination thereof. The HEM may comprise a proteinor a peptide. The HEM may be covalently attached to the staple. The HEMmay be covalently attached to the TA. The unmodified therapeutic peptidemay be selected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2,and GIP. The modified therapeutic peptide may be a derivative of apeptide selected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2,and GIP; the derivative being a peptide comprising one or more aminoacid additions, deletions, or substitutions, or a combination thereof.Non-limiting examples of mTAs include peptide lipid conjugates (PLCs).

Disclosed herein are modified therapeutic agents (mTAs) comprising atherapeutic agent (TA), a first staple, and a first HEM. The TA may be amodified or unmodified therapeutic peptide. The TA may be covalentlyattached to the first staple. The TA may be covalently attached to thefirst staple via two amino acid residues on the modified or unmodifiedtherapeutic peptide. The two amino acid residues may be at least about 4amino acid residues apart. The two amino acid residues may be at leastabout 7 amino acid residues apart. The two amino acid residues may be atleast about 11 amino acid residues apart. The two amino acids may becysteine. The half-life of the mTA may be longer than the half-life ofthe modified or unmodified therapeutic peptide alone. The first HEM maycomprise a lipid, a polyglycol region, or a combination thereof. Thelipid may be selected from a group consisting of sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, fatty amines, and fatty alcohols, and derivativesthereof. The polyglycol region comprises one or more polyethylene glycolunits, polypropylene glycol units, or polybutylene glycol units, or acombination thereof. The first HEM may comprise a protein or a peptide.The first HEM may be covalently attached to the first staple. The firstHEM may be covalently attached to the TA. The mTA may further comprise asecond staple. The mTA may further comprise a second HEM. The mTA mayfurther comprise a second staple and a second HEM. The first staple andsecond staple may be the same or different. The first HEM and second HEMmay be the same or different. The unmodified therapeutic peptide may beselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP.The modified therapeutic peptide may be a derivative of a peptideselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP;the derivative being a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof. ThemTA may be a PLC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a first HEM,wherein the therapeutic agent is a modified or unmodified therapeuticpeptide that is covalently attached to the first staple via two aminoacid residues on the modified or unmodified therapeutic peptide. The twoamino acid residues may be at least about 4 amino acid residues apart.The two amino acid residues may be at least about 7 amino acid residuesapart. The two amino acid residues may be at least about 11 amino acidresidues apart. The two amino acids may be cysteine. The half-life ofthe mTA may be longer than the half-life of the modified or unmodifiedtherapeutic peptide alone. The first HEM may comprise a lipid, apolyglycol region, or a combination thereof. The lipid may be selectedfrom a group consisting of sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, fattyamines, and fatty alcohols, and derivatives thereof. The polyglycolregion comprises one or more polyethylene glycol units, polypropyleneglycol units, or polybutylene glycol units, or a combination thereof.The first HEM may comprise a protein or a peptide. The first HEM may becovalently attached to the first staple. The first HEM may be covalentlyattached to the TA. The mTA may further comprise a second staple. ThemTA may further comprise a second HEM. The mTA may further comprise asecond staple and a second HEM. The first staple and second staple maybe the same or different. The first HEM and second HEM may be the sameor different. The unmodified therapeutic peptide may be selected fromGLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP. The modifiedtherapeutic peptide may be a derivative of a peptide selected fromGLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP; thederivative being a peptide comprising one or more amino acid additions,deletions, or substitutions, or a combination thereof. The mTA may be aPLC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a first HEM,wherein the therapeutic agent is a modified or unmodified therapeuticpeptide that is covalently attached to the first staple via two aminoacid residues on the modified or unmodified therapeutic peptide and thehalf-life of the mTA is longer than the half-life of the unmodifiedtherapeutic peptide alone. The two amino acid residues may be at leastabout 4 amino acid residues apart. The two amino acid residues may be atleast about 7 amino acid residues apart. The two amino acid residues maybe at least about 11 amino acid residues apart. The two amino acids maybe cysteine. The first HEM may comprise a lipid, a polyglycol region, ora combination thereof. The lipid may be selected from a group consistingof sterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, fatty amines, and fatty alcohols,and derivatives thereof. The polyglycol region comprises one or morepolyethylene glycol units, polypropylene glycol units, or polybutyleneglycol units, or a combination thereof. The first HEM may comprise aprotein or a peptide. The first HEM may be covalently attached to thefirst staple. The first HEM may be covalently attached to the TA. ThemTA may further comprise a second staple. The mTA may further comprise asecond HEM. The mTA may further comprise a second staple and a secondHEM. The first staple and second staple may be the same or different.The first HEM and second HEM may be the same or different. Theunmodified therapeutic peptide may be selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP. The modified therapeuticpeptide may be a derivative of a peptide selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP; the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof. The mTA may be a PLC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a first HEM,wherein the therapeutic agent is a modified or unmodified therapeuticpeptide that is covalently attached to the first staple via two aminoacid residues on the modified or unmodified therapeutic peptide and thefirst HEM is covalently attached to the first staple. The two amino acidresidues may be at least about 4 amino acid residues apart. The twoamino acid residues may be at least about 7 amino acid residues apart.The two amino acid residues may be at least about 11 amino acid residuesapart. The two amino acids may be cysteine. The half-life of the mTA maybe longer than the half-life of the modified or unmodified therapeuticpeptide alone. The first HEM may comprise a lipid, a polyglycol region,or a combination thereof. The lipid may be selected from a groupconsisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols, and derivatives thereof. The polyglycol regioncomprises one or more polyethylene glycol units, polypropylene glycolunits, or polybutylene glycol units, or a combination thereof. The firstHEM may comprise a protein or a peptide. The mTA may further comprise asecond staple. The mTA may further comprise a second HEM. The mTA mayfurther comprise a second staple and a second HEM. The first staple andsecond staple may be the same or different. The first HEM and second HEMmay be the same or different. The unmodified therapeutic peptide may beselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP.The modified therapeutic peptide may be a derivative of a peptideselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP;the derivative being a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof. ThemTA may be a PLC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a first HEM,wherein the therapeutic agent is a modified or unmodified therapeuticpeptide that is covalently attached to the first staple via two aminoacid residues on the modified or unmodified therapeutic peptide and thefirst HEM is covalently attached to the TA. The two amino acid residuesmay be at least about 4 amino acid residues apart. The two amino acidresidues may be at least about 7 amino acid residues apart. The twoamino acid residues may be at least about 11 amino acid residues apart.The two amino acids may be cysteine. The half-life of the mTA may belonger than the half-life of the modified or unmodified therapeuticpeptide alone. The first HEM may comprise a lipid, a polyglycol region,or a combination thereof. The lipid may be selected from a groupconsisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols, and derivatives thereof. The polyglycol regioncomprises one or more polyethylene glycol units, polypropylene glycolunits, or polybutylene glycol units, or a combination thereof. The firstHEM may comprise a protein or a peptide. The mTA may further comprise asecond staple. The mTA may further comprise a second HEM. The mTA mayfurther comprise a second staple and a second HEM. The first staple andsecond staple may be the same or different. The first HEM and second HEMmay be the same or different. The unmodified therapeutic peptide may beselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP.The modified therapeutic peptide may be a derivative of a peptideselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP;the derivative being a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof. ThemTA may be a PLC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a first HEM,wherein the therapeutic agent is a modified or unmodified therapeuticpeptide that is covalently attached to the first staple via two aminoacid residues on the modified or unmodified therapeutic peptide; thefirst HEM is covalently attached to the first staple; and the half-lifeof the mTA is longer than the half-life of the unmodified therapeuticpeptide alone. The two amino acid residues may be at least about 4 aminoacid residues apart. The two amino acid residues may be at least about 7amino acid residues apart. The two amino acid residues may be at leastabout 11 amino acid residues apart. The two amino acids may be cysteine.The first HEM may comprise a lipid, a polyglycol region, or acombination thereof. The lipid may be selected from a group consistingof sterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, fatty amines, and fatty alcohols,and derivatives thereof. The polyglycol region comprises one or morepolyethylene glycol units, polypropylene glycol units, or polybutyleneglycol units, or a combination thereof. The first HEM may comprise aprotein or a peptide. The mTA may further comprise a second staple. ThemTA may further comprise a second HEM. The mTA may further comprise asecond staple and a second HEM. The first staple and second staple maybe the same or different. The first HEM and second HEM may be the sameor different. The unmodified therapeutic peptide may be selected fromGLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP. The modifiedtherapeutic peptide may be a derivative of a peptide selected fromGLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP; thederivative being a peptide comprising one or more amino acid additions,deletions, or substitutions, or a combination thereof. The mTA may be aPLC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a first HEM,wherein the therapeutic agent is a modified or unmodified therapeuticpeptide that is covalently attached to the first staple via two cysteineresidues on the modified or unmodified therapeutic peptide and the firstHEM is covalently attached to the first staple. The two cysteines may beat least about 4 amino acid residues apart. The two cysteines may be atleast about 7 amino acid residues apart. The two cysteines may be atleast about 11 amino acid residues apart. The half-life of the mTA maybe longer than the half-life of the modified or unmodified therapeuticpeptide alone. The first HEM may comprise a lipid, a polyglycol region,or a combination thereof. The lipid may be selected from a groupconsisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols, and derivatives thereof. The polyglycol regioncomprises one or more polyethylene glycol units, polypropylene glycolunits, or polybutylene glycol units, or a combination thereof. The firstHEM may comprise a protein or a peptide. The mTA may further comprise asecond staple. The mTA may further comprise a second HEM. The mTA mayfurther comprise a second staple and a second HEM. The first staple andsecond staple may be the same or different. The first HEM and second HEMmay be the same or different. The unmodified therapeutic peptide may beselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP.The modified therapeutic peptide may be a derivative of a peptideselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP;the derivative being a peptide comprising one or more amino acidadditions, deletions, or substitutions, or a combination thereof. ThemTA may be a PLC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a first HEM,wherein the therapeutic agent is a modified or unmodified therapeuticpeptide that is covalently attached to the first staple via two aminoacid residues on the modified or unmodified therapeutic peptide; theunmodified therapeutic peptide is selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP; the modified therapeuticpeptide is a derivative of a peptide selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof; and the first HEM is covalentlyattached to the first staple. The two amino acid residues may be atleast about 4 amino acid residues apart. The two amino acid residues maybe at least about 7 amino acid residues apart. The two amino acidresidues may be at least about 11 amino acid residues apart. The twoamino acids may be cysteine. The half-life of the mTA may be longer thanthe half-life of the modified or unmodified therapeutic peptide alone.The first HEM may comprise a lipid, a polyglycol region, or acombination thereof. The lipid may be selected from a group consistingof sterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, fatty amines, and fatty alcohols,and derivatives thereof. The polyglycol region comprises one or morepolyethylene glycol units, polypropylene glycol units, or polybutyleneglycol units, or a combination thereof. The first HEM may comprise aprotein or a peptide. The mTA may further comprise a second staple. ThemTA may further comprise a second HEM. The mTA may further comprise asecond staple and a second HEM. The first staple and second staple maybe the same or different. The first HEM and second HEM may be the sameor different. The mTA may be a PLC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a first HEM,wherein the therapeutic agent is a modified or unmodified therapeuticpeptide that is covalently attached to the first staple via two aminoacid residues on the modified or unmodified therapeutic peptide; thefirst HEM is covalently attached to the first staple; and the first HEMcomprises a lipid, a polyglycol region, or a combination thereof. Thetwo amino acid residues may be at least about 4 amino acid residuesapart. The two amino acid residues may be at least about 7 amino acidresidues apart. The two amino acid residues may be at least about 11amino acid residues apart. The two amino acids may be cysteine. Thehalf-life of the mTA may be longer than the half-life of the modified orunmodified therapeutic peptide alone. The lipid may be selected from agroup consisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols, and derivatives thereof. The polyglycol regioncomprises one or more polyethylene glycol units, polypropylene glycolunits, or polybutylene glycol units, or a combination thereof. The firstHEM may further comprise a protein or a peptide. The mTA may furthercomprise a second staple. The mTA may further comprise a second HEM. ThemTA may further comprise a second staple and a second HEM. The firststaple and second staple may be the same or different. The first HEM andsecond HEM may be the same or different. The unmodified therapeuticpeptide may be selected from GLP-1, glucagon, oxyntomodulin, exendin-4,GLP-2, and GIP. The modified therapeutic peptide may be a derivative ofa peptide selected from GLP-1, glucagon, oxyntomodulin, exendin-4,GLP-2, and GIP; the derivative being a peptide comprising one or moreamino acid additions, deletions, or substitutions, or a combinationthereof. The mTA may be a PLC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a first HEM,wherein the therapeutic agent is a modified or unmodified therapeuticpeptide that is covalently attached to the first staple via two aminoacid residues on the modified or unmodified therapeutic peptide; theunmodified therapeutic peptide is selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP; the modified therapeuticpeptide is a derivative of a peptide selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof; the first HEM is covalentlyattached to the first staple; and the first HEM comprises a lipid, apolyglycol region, or a combination thereof. The two amino acid residuesmay be at least about 4 amino acid residues apart. The two amino acidresidues may be at least about 7 amino acid residues apart. The twoamino acid residues may be at least about 11 amino acid residues apart.The two amino acids may be cysteine. The half-life of the mTA may belonger than the half-life of the modified or unmodified therapeuticpeptide alone. The lipid may be selected from a group consisting ofsterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, fatty amines, and fatty alcohols,and derivatives thereof. The polyglycol region comprises one or morepolyethylene glycol units, polypropylene glycol units, or polybutyleneglycol units, or a combination thereof. The first HEM may furthercomprise a protein or a peptide. The mTA may further comprise a secondstaple. The mTA may further comprise a second HEM. The mTA may furthercomprise a second staple and a second HEM. The first staple and secondstaple may be the same or different. The first HEM and second HEM may bethe same or different. The mTA may be a PLC.

Further disclosed herein are modified therapeutic agents (mTAs)comprising a therapeutic agent (TA), a first staple, and a first HEM,wherein the therapeutic agent is a modified or unmodified therapeuticpeptide that is covalently attached to the first staple via two aminoacid residues on the modified or unmodified therapeutic peptide; theunmodified therapeutic peptide is selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP; the modified therapeuticpeptide is a derivative of a peptide selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof; the first HEM is covalentlyattached to the first staple; and the first HEM comprises a lipid, apolyglycol region, a peptide or protein, or a combination thereof. Thetwo amino acid residues may be at least about 4 amino acid residuesapart. The two amino acid residues may be at least about 7 amino acidresidues apart. The two amino acid residues may be at least about 11amino acid residues apart. The two amino acids may be cysteine. Thehalf-life of the mTA may be longer than the half-life of the modified orunmodified therapeutic peptide alone. The lipid may be selected from agroup consisting of sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, fatty amines,and fatty alcohols, and derivatives thereof. The polyglycol regioncomprises one or more polyethylene glycol units, polypropylene glycolunits, or polybutylene glycol units, or a combination thereof. The mTAmay further comprise a second staple. The mTA may further comprise asecond HEM. The mTA may further comprise a second staple and a secondHEM. The first staple and second staple may be the same or different.The first HEM and second HEM may be the same or different. The mTA maybe a PLC.

Further disclosed herein are modified therapeutic agents (mTAs)consisting essentially of a therapeutic agent (TA), a staple, and ahalf-life extending molecule (HEM). The TA may be a modified orunmodified therapeutic peptide. The TA may be covalently attached to thestaple. The TA may be covalently attached to the staple via two aminoacid residues on the modified or unmodified therapeutic peptide. One orboth of the two amino acid residues may be cysteine. The two amino acidresidues may be at least about 4 amino acid residues apart. The twoamino acid residues may be at least about 7 amino acid residues apart.The two amino acid residues may be at least about 11 amino acid residuesapart. The half-life of the mTA may be longer than the half-life of themodified or unmodified therapeutic peptide alone. The HEM may comprise alipid, a polyglycol region, or a combination thereof. The lipid may beselected from a group consisting of sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,fatty amines, and fatty alcohols, and derivatives thereof. Thepolyglycol region comprises one or more polyethylene glycol units,polypropylene glycol units, or polybutylene glycol units, or acombination thereof. The HEM may comprise a protein or a peptide. TheHEM may be covalently attached to the staple. The HEM may be covalentlyattached to the TA. The unmodified therapeutic peptide may be selectedfrom GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP. Themodified therapeutic peptide may be a derivative of a peptide selectedfrom GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, and GIP; thederivative being a peptide comprising one or more amino acid additions,deletions, or substitutions, or a combination thereof. Non-limitingexamples of mTAs include peptide lipid conjugates (PLCs).

In some embodiments of the mTAs disclosed herein, the HEM is directlyattached to the staple which is covalently attached to the modified orunmodified therapeutic peptide. In other embodiments of the mTAsdisclosed herein, the HEM is directly attached to the modified orunmodified therapeutic peptide. In some embodiments, HEM attachment tothe staple is preferred over HEM attachment to the modified orunmodified therapeutic peptide. In some embodiments, HEM attachment tothe modified or unmodified therapeutic peptide is preferred to HEMattachment to the staple. In some embodiments, a first mTA, wherein theHEM is directly attached to the staple which is covalently attached tothe modified or unmodified therapeutic peptide, has better activity thana second mTA, wherein the HEM is directly attached to the modified orunmodified therapeutic peptide. In other embodiments, a first mTA,wherein the HEM is directly attached to the modified or unmodifiedtherapeutic peptide, has better activity than a second mTA, wherein theHEM is directly attached to the staple which is covalently attached tothe modified or unmodified therapeutic peptide.

Half-Life Extending Molecules (HEMs)

Disclosed herein are modified therapeutic agents (mTAs) comprising atherapeutic agent (TA), a staple, and a half-life extending molecule(HEM), wherein the therapeutic agent is a modified or unmodifiedtherapeutic peptide and the half-life of the mTA is longer than thehalf-life of the modified or unmodified therapeutic peptide alone. TheHEM may be attached to the staple. The HEM may be attached to the TA.The HEM may be non-proteinaceous or proteinaceous. The HEM may comprisea lipid, a polyglycol region, a peptide or a protein, or a combinationthereof.

The HEM may be non-proteinaceous. The HEM may comprise a lipid, apolyglycol region, or a combination thereof. The HEM may be anon-proteinaceous polymer. Non-limiting examples of non-proteinaceouspolymer include hydroxyalkyl starch, such as hydroxyethyl starch (HES),polyglycol, branched polyethylene glycols, polysialic acid, polyvinylalcohol, polycarboxylate, poly(vinylpyrrolidone), dextran, or anotherbiocompatible polymer.

The lipid may be selected from a group consisting of sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, fatty amines, and fatty alcohols, and derivativesthereof. The lipid may be a sterol or sterol derivative. The lipid maybe a bile acid or derivative thereof. The lipid may be a vitamin Ederivative. The lipid may be a fatty di-acid, fatty acid, fatty amide,fatty amine, or fatty alcohol. The fatty di-acid, fatty acid, fattyamide, fatty amine, or fatty alcohol may have 5-40 carbon atoms. Thefatty di-acid, fatty acid, fatty amide, fatty amine, or fatty alcoholmay have 5-30 carbon atoms. The fatty di-acid, fatty acid, fatty amide,fatty amine, or fatty alcohol may have 5-20 carbon atoms. The fattydi-acid, fatty acid, fatty amide, fatty amine, or fatty alcohol may have6-40 carbon atoms. The fatty di-acid, fatty acid, fatty amide, fattyamine, or fatty alcohol may have 6-30 carbon atoms. The fatty di-acid,fatty acid, fatty amide, fatty amine, or fatty alcohol may have 7-40carbon atoms. The fatty di-acid, fatty acid, fatty amide, fatty amine,or fatty alcohol may have 8-40 carbon atoms. The fatty di-acid, fattyacid, fatty amide, fatty amine, or fatty alcohol may have 9-40 carbonatoms. The fatty di-acid, fatty acid, fatty amide, fatty amine, or fattyalcohol may have 10-40 carbon atoms.

The lipid may be selected from a group consisting of propanoic acid,butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoicacid, nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid,tridecanoic acid, tetradecanoic acid, myristic acid, pentadecanoic acid,hexadecanoic acid, heptadecanoic acid, octadecanoic acid, nonadecanoicacid, eicosanoic acid, heneicosanoic acid, docosanoic acid, tricosanoicacid, tetracosanoic acid, pentacosanoic acid, hexacosanoic acid,heptacosanoic acid, octacosanoic acid, nonacosanoic acid, triacontanoicacid, henatriacontanoic acid, dotriacontanoic acid, tritriacontanoicacid, tetratriacontanoic acid, pentatriacontanoic acid andhexatriacontanoic acid. The lipid may be selected from a groupconsisting of malonic acid, succinic acid, glutaric acid, adipic acid,pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioicacid, dodecanedioic acid, tridecanedioic acid, tetradecanedioic acid,pentadecanedioic acid, hexadecanedioic acid, heptadecanedioic acid,octadecanedioic acid, and nonadecanedioic acid. The lipid may beselected from a group consisting of myristoleic acid, palmitoleic acid,sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid,linoelaidic acid, α-linolenic acid, arachidonic acid, eicosapentanoicacid, erucic acid, docosahexaenoic acid. The lipid may be selected froma group consisting of cholesterol, 7-OH cholesterol,7,25-dihydroxycholesterol, cholic acid, chenodeoxycholic acid,lithocholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholicacid, glycolithocholic acid, and glycochenodeoxycholic acid.

The polyglycol region may comprise one or more polyethylene glycolunits, polypropylene glycol units, or polybutylene glycol units, or acombination thereof. The polyglycol region may comprise one or morepolyethylene glycol units. The polyglycol region may comprise one ormore polypropylene glycol units. The polyglycol region may comprise oneor more polybutylene glycol units.

The polyglycol region may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90,100, or more polyethylene glycol units, polypropylene glycol units, orpolybutylene glycol units, or a combination thereof. The polyglycolregion may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or morepolyethylene glycol units. The polyglycol region may comprise 10, 20,30, 40, 50, 60, 70, 80, 90, 100, or more polypropylene glycol units. Thepolyglycol region may comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,or more polybutylene glycol units.

The polyglycol region may comprise 100, 200, 300, 400, 500, 600, 700,800, 900, 1000, or more polyethylene glycol units, polypropylene glycolunits, or polybutylene glycol units, or a combination thereof. Thepolyglycol region may comprise 100, 200, 300, 400, 500, 600, 700, 800,900, 1000, or more polyethylene glycol units. The polyglycol region maycomprise 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or morepolypropylene glycol units. The polyglycol region may comprise 100, 200,300, 400, 500, 600, 700, 800, 900, 1000, or more polybutylene glycolunits.

The polyglycol region may comprise 1000, 2000, 3000, 4000, 5000, 6000,7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000,17000, 18000, 19000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, ormore polyethylene glycol units, polypropylene glycol units, orpolybutylene glycol units, or a combination thereof. The polyglycolregion may comprise 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000,9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000,19000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, or morepolyethylene glycol units. The polyglycol region may comprise 1000,2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000,13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 25000, 30000,35000, 40000, 45000, 50000, or more polypropylene glycol units. Thepolyglycol region may comprise 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000,18000, 19000, 20000, 25000, 30000, 35000, 40000, 45000, 50000, or morepolybutylene glycol units.

The polyglycol region may comprise a molecular weight of 500-50,000daltons. The polyglycol region may comprise a molecular weight of500-40,000 daltons. The polyglycol region may comprise a molecularweight of 500-30,000 daltons. The polyglycol region may comprise amolecular weight of 500-20,000 daltons. The polyglycol region maycomprise a molecular weight of 500, 600, 700, 800, 900, 1000, 2000,3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 15000, 20000, 25000,30000, 35000, 40000, or 45000 daltons or more, including incrementstherein.

The HEM may comprise a peptide or protein. Non-limiting examples includeserum albumin, transferrin, or the Fc domain of immunoglobulins, orvariants thereof. Variants may occur naturally or be non-naturallyoccurring. Non-naturally occurring variants may be produced usingmutagenesis techniques known in the art. Variants may comprise one ormore conservative or non-conservative amino acid substitutions,deletions, or additions, or a combination thereof. The HEM may comprisean extended recombinant polypeptide (XTEN).

Peptide Lipid Conjugate (PLC)

Disclosed herein are peptide lipid conjugates (PLCs) comprising one ormore lipids attached to one or more peptide conjugates, the peptideconjugate (PC) comprising (a) one or more peptide regions comprisingtherapeutic agents (TAs); and (b) one or more staples, the one or morestaples connecting two or more residues in the peptide region. The lipidconjugate may further comprise one or more polyethylene glycol subunits.The one or more lipids may be pegylated. The one or more lipids may beconjugated to the one or more therapeutic agents. The one or more lipidsmay be conjugated to the one or more staples. At least one of the two ormore residues in the peptide region may be cysteine. The two or moreresidues in the peptide region may comprise cysteine. The two or moreresidues may be at least about 4 amino acids apart. The two or moreresidues may be at least about 7 amino acids apart. The two or moreresidues may be at least about 11 amino acids apart.

FIG. 2A-G depict schematics of exemplary peptide lipid conjugates. FIG.2A depicts a peptide lipid conjugate comprising a peptide conjugate (PC)attached to a lipid (L). FIG. 2B depicts a peptide lipid conjugatecomprising (a) a peptide conjugate comprising a staple (C) attached totwo cysteine residues (*) on a peptide therapeutic agent (TA); and (b) alipid (L), wherein the lipid is attached to the staple. FIG. 2C depictsa peptide lipid conjugate comprising (a) a peptide conjugate comprisinga staple (C) attached to two cysteine residues (*) on a peptidetherapeutic agent (TA); and (b) a lipid (L), wherein the lipid isattached to the TA. FIG. 2D depicts a peptide lipid conjugate comprising(a) a peptide conjugate comprising a staple (C) attached to two cysteineresidues (*) on two peptide therapeutic agents (TA¹ and TA²); and (b) alipid (L), wherein the lipid is attached to the staple. FIG. 2E depictsa peptide lipid conjugate comprising (a) a peptide conjugate comprisinga staple (C) attached to two cysteine residues (*) on two peptidetherapeutic agents (TA¹ and TA²); and (b) a lipid (L), wherein the lipidis attached to the peptide therapeutic agent (TA¹). FIG. 2F depicts apeptide lipid conjugate comprising (a) a peptide conjugate comprising astaple (C) attached to two cysteine residues on a peptide therapeuticagent (TA); and (b) a pegylated lipid (L), wherein the lipid is attachedto the TA. FIG. 2G depicts a peptide lipid conjugate comprising (a) apeptide conjugate comprising a staple (C) attached to two cysteineresidues on a peptide therapeutic agent (TA); and (b) a lipid (L),wherein the lipid is attached to the TA. The lipid may be attached to anamino acid residue within the therapeutic agent. The amino acid residuemay be a cysteine residue. Alternatively, the amino acid residue is nota cysteine residue.

Additional exemplary peptide lipid conjugates are depicted in FIGS.4A-H, 5A-H and 6A-H. FIG. 4A-C depict a peptide lipid conjugatecomprising (a) a peptide conjugate comprising a staple (C) attached to atherapeutic agent (TA); and (b) a lipid (L). The TA may be a protein.The lipid may be attached to any portion of the TA. For example, asshown in FIG. 4A, the lipid may be attached to the C-terminus of atherapeutic agent comprising a protein. As shown in FIG. 4B, the lipidmay be attached to an internal region of a therapeutic agent comprisinga protein. As shown in FIG. 4C, the lipid may be attached to theN-terminus of a therapeutic agent comprising a protein. FIG. 4D-F depicta peptide lipid conjugate comprising (a) a peptide conjugate comprisinga staple (C) attached to a therapeutic agent (TA); and (b) two lipids(L¹ and L²). The two lipids may be attached to any portion of the TA.The two lipids may be attached to one or more ends of the TA. The twolipids may be attached to an internal region of the TA. The two lipidsmay be attached to an end of the TA and to an internal region of the T.As shown in FIG. 4D, the first lipid (L¹) is attached to an internalregion of the TA and the second lipid (L²) is attached to one end of theTA. As shown in FIG. 4E, the first lipid (L¹) and the second lipid (L²)are attached to opposite ends of the TA. As shown in FIG. 4F, the firstlipid (L¹) is attached to one end of the TA and the second lipid (L²) isattached to an internal region of the TA. The PLCs disclosed herein maycomprise (a) a peptide conjugate comprising a staple (C) and atherapeutic agent (TA); and (b) a plurality of lipids (L¹ . . . L^(n)).FIG. 4G depicts a PLC comprising (a) a peptide conjugate comprising astaple (C) attached to a therapeutic agent (TA); and (b) three lipids(L¹, L² and L³). As shown in FIG. 4G, all three lipids are attached tovarious regions within the TA. FIG. 4H depicts a PLC comprising (a) apeptide conjugate comprising a staple (C) attached to a therapeuticagent (TA); and (b) four lipids (L¹, L², L³ and L⁴). As shown in FIG.4H, all four lipids are attached to various regions within the TA.

FIG. 5A-C depict a peptide lipid conjugate comprising (a) a peptideconjugate comprising a staple (C) attached to a therapeutic agent (TA);and (b) a lipid (L). The lipid may be attached to any portion of thestaple (C). For example, as shown in FIGS. 5A and 5C, the lipid may beattached to one end of the staple (C). As shown in FIG. 5B, the lipidmay be attached to an internal region of the staple. FIG. 5D-F depict apeptide lipid conjugate comprising (a) a peptide conjugate comprising astaple (C) attached to a therapeutic agent (TA); and (b) two lipids (L¹and L²). The two lipids may be attached to any portion of the staple.The two lipids may be attached to one or more ends of the staple. Thetwo lipids may be attached to an internal region of the staple. The twolipids may be attached to an end of the staple and to an internal regionof the staple. As shown in FIG. 5D, the first lipid (L¹) is attached toone end of the TA and the second lipid (L²) is attached to an internalregion of the TA. As shown in FIG. 5E, the first lipid (L¹) and thesecond lipid (L²) are attached to opposite ends of the staple. As shownin FIG. 5F, the first lipid (L¹) is attached to an internal region ofthe staple and the second lipid (L²) is attached to one end of thestaple. The PLCs disclosed herein may comprise (a) a peptide conjugatecomprising a staple (C) and a therapeutic agent (TA); and (b) aplurality of lipids (L¹ . . . L^(n)). FIG. 5G depicts a PLC comprising(a) a peptide conjugate comprising a staple (C) attached to atherapeutic agent (TA); and (b) three lipids (L¹, L² and L³). As shownin FIG. 5G, all three lipids are attached to various regions within thestaple. FIG. 5H depicts a PLC comprising (a) a peptide conjugatecomprising a staple (C) attached to a therapeutic agent (TA); and (b)four lipids (L¹, L², L³ and L⁴). As shown in FIG. 5H, all four lipidsare attached to various regions within the staple.

FIG. 6A-C depict a peptide lipid conjugate comprising (a) a peptideconjugate comprising a staple (C) attached to a therapeutic agent (TA);and (b) two lipids (L¹ and L²). The two lipids may be attached to anyportion of the TA, staple (C), or a combination thereof. At least onelipid may be attached to any portion of the TA. At least one lipid maybe attached to any portion of the staple. The two lipids may be attachedto any portion of the TA and the staple. For example, as shown in FIG.6A, the first lipid (L¹) is attached to an internal region of the stapleand the second lipid (L²) is attached to one end of the therapeuticagent. As shown in FIG. 6B, the first lipid (L¹) is attached to aninternal region of the staple and the second lipid (L²) is attached toan internal region of the therapeutic agent. As shown in FIG. 6C, thefirst lipid (L¹) is attached to one end of the staple and the secondlipid (L²) is attached to one end of the therapeutic agent. FIG. 6D-Hdepict a peptide lipid conjugate comprising (a) a peptide conjugatecomprising a staple (C) attached to a therapeutic agent (TA); and (b) aplurality of lipids (L¹ . . . L^(n)). The plurality of lipids may beattached to any portion of the TA. The plurality of lipids may beattached to one or more ends of the TA. The plurality of lipids may beattached to an internal region of the TA. The plurality of lipids may beattached to any portion of the staple. The plurality of lipids may beattached to one or more ends of the staple. The plurality of lipids maybe attached to an internal region of the staple. The plurality of lipidsmay be attached to one or more ends of the TA, one or more internalregions of the TA, one or more ends of the staple (C), one or moreinternal regions of the staple (C), or a combination thereof. FIG. 6Ddepicts a peptide lipid conjugate comprising (a) a peptide conjugatecomprising a therapeutic agent (TA) and a staple (C); and (b) threelipids (L¹, L² and L³). As shown in FIG. 6D, the first lipid (L¹) isattached to one end of the staple (C), the second lipid (L²) is attachedto an internal region of the TA and the third lipid (L³) is attached toone end of the TA. FIG. 6E-F depict a peptide lipid conjugate comprising(a) a peptide conjugate comprising a therapeutic agent (TA) and a staple(C); and (b) four lipids (L¹, L², L³ and L⁴). As shown in FIG. 6E, thefirst lipid (L¹) and the second lipid (L²) are attached to opposite endsof the staple (C) and the third lipid (L³) and fourth lipid (L⁴) areattached to opposite ends of the TA. As shown in FIG. 6F, the firstlipid (L¹) is attached to one end of the staple (C); the second lipid(L²) is attached to an internal region of the staple (C); the thirdlipid (L³) is attached to one end of the TA and the fourth lipid (L⁴) isattached to an internal region of the TA. As shown in FIG. 6G, at leastone lipid (L¹) is attached to the staple (C) and three lipids (L², L³and L⁴) are attached to various regions within the TA. As shown in FIG.6H, at least two lipids ((L¹ and L²) are attached to the staple and fourlipids (L³, L⁴, L⁵ and L⁶) are attached to various regions within theTA.

The PLCs disclosed herein may have the structure:PC-A¹-P¹-L   Formula (II)wherein:PC is the peptide conjugate;A¹ is a chemical group linking PC and P¹;P¹ is a bond or -PEG-A²-;PEG is a chemical group comprising one or more polyethylene glycolsubunits;A² is a chemical group linking PEG and L; andL is the lipid.

The PEG of Formula (II) may be selected from:

whereinm and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20.

In some embodiments of a PLC of Formula (II) disclosed herein,

A¹ is selected from

each R¹, R², R³, and R⁴ are independently selected from H, halo, CN,—SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;each R⁵ is independently selected from H, alkyl, haloalkyl, arylalkyl,and heteroalkyl;k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;p is 2, 3, 4, 5, 6, 7, 8, 9, or 10; andq is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments described of a PLC of Formula (II) disclosed herein,

A² is selected from a bond,

X is a bond, NR⁵, S, or O;R¹, R², R³, and R⁴ are independently selected from H, halo, CN, —SR⁵,alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;R⁵ is H, alkyl, haloalkyl, arylalkyl, or heteroalkyl;R⁶ is H, alkyl, arylalkyl, —(CR¹R²)_(t)SR⁵, —(CR¹R²)_(t)NR⁵R⁵,—(CR¹R²)_(t)OR⁵, or —(CR¹R²)_(t)CO₂R⁵;each R⁷ is independently selected from H, alkyl, haloalkyl, arylalkyl,and heteroalkyl;r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;s is 1, 2, 3, 4, or 5; andt is 0, 1, 2, 3, 4, or 5.

In some embodiments described of a PLC of Formula (II) disclosed herein,P¹ is -PEG-A².

Disclosed herein are methods of producing a PLC of Formula (II), themethod comprising reacting an amino acid residue of TA with A³-P¹-L,wherein A³ is a reactive precursor to form A¹. A³ may be ahaloacetamide, maleimide, benzyl halide, alkyl disulfide, or pyridyldisulfide. A³ may be a haloacetamide. A³ may be a bromoacetamide. A³ maybe an alkyl disulfide.

The peptide lipid conjugates (PLCs) may comprise (a) one or more lipids,the lipids selected from a group consisting of sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, and fatty alcohols; and (b) one or more peptideconjugates (PC), the peptide conjugate comprising a peptide regioncomprising one or more peptide therapeutic agents (TA) and a stapleregion comprising one or more staples, the one or more staples connecttwo or more residues in the peptide region, wherein the one or morelipids are attached to the one or more peptide conjugates. The one ormore lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. The one or morelipids may be attached to one or more ends of the TA, to an internalregion of the TA, to one or more ends of the staple, to an internalregion of the staple, or a combination thereof. The one or more lipidsmay be attached to both the TA and the staple. At least one of the twoor more residues may be cysteine. The two or more residues in thepeptide region may comprise cysteine. In some instances, at least one ofthe two or more residues is not cysteine. The two or more residues maybe at least about 4 amino acid residues apart. The two or more residuesmay be at least about 7 or more residues apart. The two or more residuesmay be at least about 11 or more residues apart.

The peptide lipid conjugates (PLCs) may comprise (a) one or more lipids;and (b) one or more peptide conjugates (PC), wherein the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, the one or more peptide therapeutic agents comprisingone or more oxyntomodulin, exenatide, exendin-4, glucagon-like protein-1(GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R andGCGR dual agonist, or a derivative thereof, wherein the one or morelipids are attached to the one or more peptide conjugates. The one ormore lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. The one or morelipids may be attached to one or more ends of the TA, to an internalregion of the TA, to one or more ends of the staple, to an internalregion of the staple, or a combination thereof. The one or more lipidsmay be attached to both the TA and the staple. At least one of the twoor more residues may be cysteine. The two or more residues in thepeptide region may comprise cysteine. In some instances, at least one ofthe two or more residues is not cysteine. The two or more residues maybe at least about 4 amino acid residues apart. The two or more residuesmay be at least about 7 or more residues apart. The two or more residuesmay be at least about 11 or more residues apart.

The peptide lipid conjugates (PLCs) may comprise (a) one or more lipids;and (b) one or more peptide conjugates (PC), wherein the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, the one or more peptide therapeutic agents comprisingGLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, GIP, GLP1R and GCGRdual agonist, GLP1R and GIPR dual agonist, or GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof, wherein the one or more lipids areattached to the one or more peptide conjugates. The one or more lipidsmay be attached to the one or more TAs. The one or more lipids may beattached to the one or more staples. The one or more lipids may beattached to one or more ends of the TA, to an internal region of the TA,to one or more ends of the staple, to an internal region of the staple,or a combination thereof. The one or more lipids may be attached to boththe TA and the staple. At least one of the two or more residues may becysteine. The two or more residues in the peptide region may comprisecysteine. In some instances, at least one of the two or more residues isnot cysteine. The two or more residues may be at least about 4 aminoacid residues apart. The two or more residues may be at least about 7 ormore residues apart. The two or more residues may be at least about 11or more residues apart.

The PLCs disclosed herein may have the structure:

wherein:TA is the therapeutic agent;each Q is the same or different, and is a staple;each A¹ is the same or different, and is a chemical group linking Q andP¹;each P¹ is a bond or -PEG-A²-;each PEG is the same or different, and is a chemical group comprisingone or more polyethylene glycol subunits;each A² is the same or different, and is a chemical group linking PEGand L;each L is the same or different, and is a lipid;a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; andb is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The PEG of Formula (III) may be selected from:

whereinm and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20.

In some embodiments of a PLC of Formula (III) disclosed herein,

A¹ is selected from

each R¹, R², R³, and R⁴ are independently selected from H, halo, CN,—SR⁵, alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;each R⁵ is independently selected from H, alkyl, haloalkyl, arylalkyl,and heteroalkyl;k is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;p is 2, 3, 4, 5, 6, 7, 8, 9, or 10; andq is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments described of a PLC of Formula (III) disclosedherein,

A² is selected from a bond,

X is a bond, NR⁵, S, or O;R¹, R², R³, and R⁴ are independently selected from H, halo, CN, —SR⁵,alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;R⁵ is H, alkyl, haloalkyl, arylalkyl, or heteroalkyl;R⁶ is H, alkyl, arylalkyl, —(CR¹R²)_(t)SR, —(CR¹R²)_(t)NR⁵R⁵,—(CR¹R²)_(t)OR⁵, or —(CR¹R²)_(t)CO₂R⁵;each R⁷ is independently selected from H, alkyl, haloalkyl, arylalkyl,and heteroalkyl;r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;s is 1, 2, 3, 4, or 5; andt is 0, 1, 2, 3, 4, or 5.

In some embodiments described of a PLC of Formula (III) disclosedherein, P¹ is -PEG-A².

Disclosed herein are methods of producing a PLC of Formula (III), themethod comprising reacting one or more staples with A³-P¹-L, wherein A³is a reactive precursor to form A¹. A³ may be a haloacetamide,maleimide, benzyl halide, alkyl disulfide, or pyridyl disulfide. A³ maybe a haloacetamide. A³ may a bromoacetamide. A³ may be an alkyldisulfide.

The peptide lipid conjugates (PLCs) may comprise (a) one or more lipids,the lipids selected from a group consisting of sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, and fatty alcohols; and (b) one or more peptideconjugates (PC), the peptide conjugate comprising a peptide regioncomprising one or more peptide therapeutic agents (TA) and a stapleregion comprising one or more staples, the one or more staples connecttwo or more residues in the peptide region, wherein the one or morelipids are attached to the one or more staples. The two or more residuesin the peptide region may comprise cysteine.

The peptide lipid conjugates (PLCs) may comprise (a) one or more lipids;and (b) one or more peptide conjugates (PC), wherein the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, the one or more peptide therapeutic agents comprisingone or more oxyntomodulin, exenatide, exendin-4, glucagon-like protein-1(GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R andGCGR dual agonist, or a derivative thereof, wherein the one or morelipids are attached to the one or more staples. The two or more residuesin the peptide region may comprise cysteine.

The peptide lipid conjugates (PLCs) may comprise (a) one or more lipids;and (b) one or more peptide conjugates (PC), wherein the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, the one or more peptide therapeutic agents comprisingGLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, GIP, GLP1R and GCGRdual agonist, GLP1R and GIPR dual agonist, or GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof, wherein the one or more lipids areattached to the one or more staples. The two or more residues in thepeptide region may comprise cysteine.

The PLCs disclosed herein may have the structure:

wherein:

-   TA is a therapeutic agent;-   each Q is the same or different, and is a staple;-   each A¹ is the same or different, and is a chemical group linking Q    and P¹;-   each P¹ is a bond or -PEG-A²-;-   each PEG is the same or different, and is a chemical group    comprising one or more polyethylene glycol subunits;-   each A² is the same or different, and is a chemical group linking    PEG and L;-   each L is the same or different, and is a lipid;-   a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;-   b is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and-   c is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The peptide lipid conjugates (PLCs) may comprise (a) two or more lipids,the lipids selected from a group consisting of sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, and fatty alcohols; and (b) one or more peptideconjugates (PC), the peptide conjugate comprising a peptide regioncomprising one or more peptide therapeutic agents (TA) and a stapleregion comprising one or more staples, the one or more staples connecttwo or more residues in the peptide region, wherein at least one of thetwo or more lipids are attached to the one or more therapeutic agentsand at least one of the two or more lipids are attached to the one ormore staples. The one or more lipids may be attached to one or more endsof the TA, to an internal region of the TA, to one or more ends of thestaple, to an internal region of the staple, or a combination thereof.At least one or more lipids may be attached to one or more ends of theTA, to an internal region of the TA, to one or more ends of the staple,to an internal region of the staple, or a combination thereof. At leastone of the two or more lipids may be attached to both the TA and thestaple. At least one of the two or more residues may be cysteine. Thetwo or more residues in the peptide region may comprise cysteine. Insome instances, at least one of the two or more residues is notcysteine. The two or more residues may be at least about 4 amino acidresidues apart. The two or more residues may be at least about 7 or moreresidues apart. The two or more residues may be at least about 11 ormore residues apart.

The peptide lipid conjugates (PLCs) may comprise (a) two or more lipids;and (b) one or more peptide conjugates (PC), wherein the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, the one or more peptide therapeutic agents comprisingone or more oxyntomodulin, exenatide, exendin-4, glucagon-like protein-1(GLP-1), GLP-2, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R andGCGR dual agonist, or a derivative thereof, wherein at least one of thetwo or more lipids are attached to the one or more therapeutic agentsand at least one of the two or more lipids are attached to the one ormore staples. At least one or more lipids may be attached to one or moreends of the TA, to an internal region of the TA, to one or more ends ofthe staple, to an internal region of the staple, or a combinationthereof. At least one of the two or more lipids may be attached to boththe TA and the staple. At least one of the two or more residues may becysteine. The two or more residues in the peptide region may comprisecysteine. In some instances, at least one of the two or more residues isnot cysteine. The two or more residues may be at least about 4 aminoacid residues apart. The two or more residues may be at least about 7 ormore residues apart. The two or more residues may be at least about 11or more residues apart.

The peptide lipid conjugates (PLCs) may comprise (a) two or more lipids;and (b) one or more peptide conjugates (PC), wherein the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, the one or more peptide therapeutic agents comprisingGLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, GIP, GLP1R and GCGRdual agonist, GLP1R and GIPR dual agonist, or GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof, wherein at least one of the two ormore lipids are attached to the one or more therapeutic agents and atleast one of the two or more lipids are attached to the one or morestaples. At least one or more lipids may be attached to one or more endsof the TA, to an internal region of the TA, to one or more ends of thestaple, to an internal region of the staple, or a combination thereof.At least one of the two or more lipids may be attached to both the TAand the staple. At least one of the two or more residues may becysteine. The two or more residues in the peptide region may comprisecysteine. In some instances, at least one of the two or more residues isnot cysteine. The two or more residues may be at least about 4 aminoacid residues apart. The two or more residues may be at least about 7 ormore residues apart. The two or more residues may be at least about 11or more residues apart.

Lipids

The mTAs or PLCs disclosed herein may comprise one or more lipids. Thelipid may be attached to one or more peptide conjugates. The attachmentof the one or more lipids to the one or more peptide conjugates maycomprise a covalent attachment. The lipid may be attached to one or moreTAs. The lipid may be attached to one or more staples. The lipid may beattached to the peptide conjugate, TA, or staple via one or more aminoacid residues. The one or more amino acid residues may comprise acysteine residue. Alternatively, the one or more amino acid residues donot comprise a cysteine residue. The lipid may be attached to thepeptide conjugate or lipid via one or more functional groups. The one ormore functional groups may comprise a ketone. The one or more functionalgroups may comprise a carbonyl. Attachment of the lipid to the peptideconjugate, TA, or staple may enhance one or more pharmacokineticproperties of the TAs.

The one or more lipids may be fatty acids. Fatty acids may be fattydi-acids, fatty amines, fatty amides or fatty alcohols. Fatty acids maybe saturated or unsaturated Saturated fatty acids include, but are notlimited to, lauric acid, myristic acid, palmitic acid, stearic acid,arachidic acid. Unsaturated fatty acids include, but are not limited topalmitoleic acid, oleic acid, linoleic acid, linolenic acid, erucic acidand arachidonic acid. Fatty acids may be short-chain fatty acids, mediumchain fatty acids, long chain fatty acids or very long chain fattyacids. Fatty acids may be monounsaturated or polyunsaturated. Fattyacids may be omega fatty acids, essential fatty acids, partiallyhydrogenated fatty acids, cis-isomer fatty acids, or trans-isomer fattyacids. Fatty acids may be omega-3 fatty acids, omega-6 fatty acids oromega-9 fatty acids.

The fatty acid may comprise a chain of about 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more carbon atoms.The fatty acid may comprise a chain of 6-40 carbon atoms. The fatty acidmay comprise a chain of 7-40 carbon atoms. The fatty acid may comprise achain of 8-40 carbon atoms. The fatty acid may comprise a chain of 9-40carbon atoms. The fatty acid may comprise a chain of 10-40 carbon atoms.The fatty acid may comprise a carbon chain further comprising 1, 2, 3,4, 5, 6 or more double bonds. The fatty acid may be naturally occurring.The fatty acid may not be naturally occurring. The fatty acid may besynthesized.

The PLCs or mTAs disclosed herein may further comprise one or more fattyacids. The PLCs or mTAs disclosed herein may further comprise two ormore fatty acids. The PLCs or mTAs disclosed herein may further comprisethree or more fatty acids. The PLCs or mTAs disclosed herein may furthercomprise four or more fatty acids. The PLCs or mTAs disclosed herein mayfurther comprise five or more fatty acids. The fatty acids may bedifferent. The fatty acids may be the same.

The one or more lipids of the PLC or mTA may be selected from the groupconsisting of myristic acid, docosahexanoic acid, lithocholic acidester, cholic acid and palmitic acid. The one or more lipids of the PLCor mTA may be myristic acid. The one or more lipids of the PLC or mTAmay be docosahexanoic acid. The one or more lipids of the PLC or mTA maybe lithocholic acid ester. The one or more lipids of the PLC or mTA maybe cholic acid. The one or more lipids of the PLC or mTA may be palmiticacid.

The PLCs or mTAs may comprise one or more sterols or sterol derivatives.The sterols or sterol derivatives may be selected from a groupcomprising cholesterol, 7-OH cholesterol, 7,25-dihydroxycholesterol,cholic acid, chenodeoxycholic acid, lithocholic acid, deoxycholic acid,glycocholic acid, glycodeoxycholic acid, glycolithocholic acid, andglycochenodeoxycholic acid.

The PLCs or mTAs may comprise one or more bile acids. The bile acids maybe selected from a group comprising cholic acid, chenodeoxycholic acid,lithocholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholicacid, glycolithocholic acid, and glycochenodeoxycholic acid.

The PLC or mTA may comprise one or more Vitamin E derivatives. TheVitamin E derivatives may be selected from a group comprisingα-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol,β-tocotrienol, γ-tocotrienol and δ-tocotrienol.

Pegylated Lipid

The PLCs disclosed herein may comprise one or more pegylated lipids. ThemTAs disclosed herein may comprise one or more pegylated lipids.

The pegylated lipid may be attached to the therapeutic agent. Thepegylated lipid may be attached to any portion of the therapeutic agent.For example, the pegylated lipid may be attached to one or more ends ofthe therapeutic agent. The pegylated lipid may be attached to one ormore internal regions of the therapeutic agent.

The pegylated lipid may be attached to one or more peptide conjugates.The pegylated lipid may be attached to one or more TAs. The pegylatedlipid may be attached to one or more staples. The pegylated lipid may beattached to the peptide conjugate, TA, or staple via one or more aminoacid residues. The one or more amino acid residues may comprise acysteine residue. Alternatively, the one or more amino acid residues donot comprise a cysteine residue. The pegylated lipid may be attached tothe staple, peptide conjugate or pegylated lipid via one or morefunctional groups. The one or more functional groups may comprise aketone. The one or more functional groups may comprise a carbonyl.Attachment of the pegylated lipid to the peptide conjugate, TA, orstaple may enhance one or more pharmacokinetic properties of the TAs.

A pegylated lipid may comprise at least one polyethylene glycol subunit.The connection between the lipid and the one or more polyethylene glycolsubunits may be a direct bond or a linker (A²). Non-limiting examples ofa linker between the lipid and the one or more polyethylene glycolsubunits include:

whereinX is a bond, NR⁵, S, or O;R¹, R², R³, and R⁴ are independently selected from H, halo, CN, —SR⁵,alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;R⁵ is H, alkyl, haloalkyl, arylalkyl, or heteroalkyl;R⁶ is H, alkyl, arylalkyl, —(CR¹R²)_(t)SR⁵, —(CR¹R²)_(t)NR⁵R⁵,—(CR¹R²)_(t)OR⁵, or —(CR¹R²)_(t)CO₂R⁵;each R⁷ is independently selected from H, alkyl, haloalkyl, arylalkyl,and heteroalkyl;r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;s is 1, 2, 3, 4, or 5; andt is 0, 1, 2, 3, 4, or 5.

A pegylated lipid may have the structure P¹-L, wherein P¹ is -PEG-A²-;PEG is a chemical group comprising one or more polyethylene glycolsubunits; A² is a chemical group linking PEG and L; and L is a lipid.PEG may be selected from:

whereinm and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20.

A pegylated lipid may be connected to a therapeutic agent through alinker. A pegylated lipid may be connected to a staple through a linker.

Lipid Derivatives

The lipid derivatives may be directly attached to a TA or a staple. Suchattachment of the lipid derivative to the TAs may enhance thepharmacokinetic properties of the TAs. The mTAs and PLCs disclosedherein may comprise one or more lipid derivatives.

The lipid derivatives may be attached to the therapeutic agent. Thelipid derivatives may be attached to any portion of the therapeuticagent. For example, lipid derivatives may be attached to one or moreends of the therapeutic agent. The lipid derivatives may be attached toone or more internal regions of the therapeutic agent.

The lipid derivatives may be attached to a peptide conjugate. The lipidderivatives may be attached to a TA. The lipid derivatives may beattached to a staple. The lipid derivative may be attached to thepeptide conjugate, TA, or staple via one or more amino acid residues.The lipid derivative may be attached to one or more ends and/or internalregions of the peptide conjugate. The lipid derivative may be attachedto one or more ends and/or internal regions of the therapeutic agent.The lipid derivative may be attached to one or more ends and/or internalregions of the staple. The lipid derivative may be attached to one ormore ends and/or internal regions of the peptide conjugate, TA, staple,or a combination thereof. The one or more amino acid residues maycomprise a cysteine residue. Alternatively, the one or more amino acidresidues do not comprise a cysteine residue. The lipid derivative may beattached to the peptide conjugate or lipid via one or more functionalgroups. The one or more functional groups may comprise a ketone. The oneor more functional groups may comprise a carbonyl. Attachment of thelipid derivative to the peptide conjugate, TA or staple may enhance thepharmacokinetic properties of the TAs.

Lipid derivatives may be pegylated. A pegylated lipid may comprise atleast one polyethylene glycol subunit. The lipid derivatives may be notpegylated. Lipids may be broadly defined as hydrophobic or amphiphilicsmall molecules. Lipids may be naturally occurring or synthetic. Lipidsmay be eicosanoids, prostaglandins, leukotrienes, thromboxanes, waxesters, coenzyme A derivatives, fatty acid carnitines, fatty acidamides, ethanolamines, bile acids, vitamin E, vitamin A, vitamin D,vitamin K, fat-soluble vitamin derivatives, monoglycerides,diglycerides, triglycerides, phospholipids, phosphatidylcholine,glycerolipids, glycerols, glycerophospholipids, sphingolipids,saccharolipids, polyketides, sterols, sterol derivatives, sterol lipids,steroid hormones, prenol lipids, carotenoids, fatty acids, and fattyalcohols.

In one aspect, disclosed herein are lipid derivatives having thestructure of A³-P¹-L, wherein:

A³ is a haloacetamide, maleimide, benzyl halide, alkyl disulfide, orpyridyl disulfide;

P¹ is a bond or -PEG-A²-;

PEG is a chemical group comprising one or more polyethylene glycolsubunits;

A² is a chemical group linking PEG and L; and

L is a lipid selected from sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols.

In some embodiments described herein, PEG is selected from:

whereinm and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, or 20.

In some embodiments described herein,

A² is selected from a bond,

X is a bond, NR⁵, S, or O;R¹, R², R³, and R⁴ are independently selected from H, halo, CN, —SR⁵,alkyl, cycloalkyl, haloalkyl, —NR⁵R⁵, and —OR⁵;R⁵ is H, alkyl, haloalkyl, arylalkyl, or heteroalkyl;R⁶ is H, alkyl, arylalkyl, —(CR¹R²)_(t)SR⁵, —(CR¹R²)_(t)NR⁵R⁵,—(CR¹R²)_(t)OR⁵, or —(CR¹R²)_(t)CO₂R⁵;each R⁷ is independently selected from H, alkyl, haloalkyl, arylalkyl,and heteroalkyl;r is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;s is 1, 2, 3, 4, or 5; andt is 0, 1, 2, 3, 4, or 5.

Reaction of a lipid derivative with a staple precursor compound mayproduce a lipid staple precursor. The lipid staple precursor maysubsequently be reacted with one or more therapeutic agents to form aPLC or mTA. Alternatively, reaction of a lipid derivative with aderivatizable functional group of the staple already attached to one ormore therapeutic agents produces the PLC or mTA. Similarly, reaction ofa lipid derivative with an amino acid on the TA produces an PLC or mTA.

Peptide Conjugate

The PLCs or mTAs disclosed herein may comprise one or more peptideconjugates. The peptide conjugates may comprise one or more staplesconnected two or more amino acid residues on one or more therapeuticagents. The peptide conjugates may further comprise one or moreadditional staples. The one or more additional staples may be attachedto one or more amino acid residues on the one or more therapeuticagents. The one or more amino acid residues may comprise a cysteineresidue on the one or more therapeutic agents. Alternatively, the one ormore amino acid residues do not comprise a cysteine residue on the oneor more therapeutic agents. The one or more additional staples may beattached to the one or more staples.

The peptide conjugates may comprise one or more staples connected one ormore therapeutic agents, wherein at least two residues on the one ormore therapeutic agents are connected to the one or more staples. Thetwo residues may be on the same therapeutic agent. The two residues maybe on different therapeutic agents. The two residues may be connected tothe same staple. The two residues may be connected to different staples.Additional therapeutic agents may be attached to the one or moretherapeutic agents or one or more staples. Attachment of the additionaltherapeutic agents to the one or more staples may occur via one or moreamino acid residues. The one or more amino acid residues may comprise acysteine residue. Alternatively, the one or more amino acid residues donot comprise a cysteine residue. Additional staples may be attached tothe one or more therapeutic agents or one or more staples. Attachment ofthe additional staples to the one or more therapeutic agents may occurvia one or more amino acid residues. The one or more amino acid residuesmay comprise a cysteine residue. Alternatively, the one or more aminoacid residues do not comprise a cysteine residue.

FIG. 1A depicts a peptide conjugate comprising a staple (C) connected totwo cysteine residues (*) in a single peptide therapeutic agent (TA).FIG. 1B depicts a peptide conjugate comprising a staple (C) connected totwo cysteine residues (*) in two peptide therapeutic agents (TA¹ andTA²). FIG. 1C depicts a peptide agent comprising two staples (C¹ and C²)and two peptide therapeutic agents (TA¹ and TA²), wherein C¹ connects toa cysteine residue (*) in TA¹ and C² connects to a cysteine residue (*)in TA² and C¹ and C² are connected to each other. FIG. 1D depicts apeptide agent comprising two staples (C¹ and C²) and two peptidetherapeutic agents (TA¹ and TA²), wherein C¹ connects to a cysteineresidue (*) in TA¹ and a cysteine residue (*) in TA² and C² is connectedto TA¹. FIG. 1E depicts a peptide agent comprising two staples (C¹ andC²) and a peptide therapeutic agent (TA¹), wherein C1 connects to acysteine residue (*) in TA and C² connects to a cysteine residue (*) inTA and C¹ and C² are connected to each other. FIG. 1F depicts a peptideconjugate comprising a staple (C) connected to two cysteine residues ina single peptide therapeutic agent (TA). The one or more staples may beattached to an amino acid in the peptide therapeutic agent. The aminoacid residue may be a cysteine residue. Alternatively, the amino acidresidue is not a cysteine residue.

Staple

The PLCs or mTAs disclosed herein may comprise one or more staples. ThePLCs or mTAs disclosed herein may comprise two or more staples. The PLCsor mTAs disclosed herein may comprise three or more staples. The PLCs ormTAs disclosed herein may comprise four or more staples. The PLCs ormTAs disclosed herein may comprise five or more staples. The PLCs ormTAs disclosed herein may comprise 6, 7, 8, 9, 10 or more staples.

The one or more staples may connect two or more amino acid residues in apeptide region of a peptide conjugate. At least one of the two or moreamino acid residues may be a cysteine residue. The two or more aminoacid residues may both be cysteine residues. The one or more staples mayconnect two or more cysteine residues on the same TA. The one or morestaples may connect two or more cysteine residues on two or more TAs.Two or more staples may connect two or more cysteine residues on thesame TA. Two or more staples may connect two or more cysteine residueson two or more TAs. In some instances, at least one of the two or moreamino acid residues are not cysteine. In some instances, at least two ofthe two or more amino acid residues are not cysteine. At least one ofthe two or more amino acid residues may be a cysteine residue. The oneor more staples may connect two or more amino acid residues on the sameTA. The one or more staples may connect two or more amino acid residueson two or more TAs. Two or more staples may connect two or more aminoacid residues on the same TA. Two or more staples may connect two ormore amino acid residues on two or more TAs. The two or more TAs may bethe same. The two or more TAs may be different.

At least one staple may connect at least two amino acid residues on atherapeutic agent. The two amino acid residues may be adjacent. The twoamino acid residues may be non-adjacent. The two amino acid residues maybe at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more amino acid residues apart. The two amino acidresidues may be at least about 4 amino acid residues apart. The twoamino acid residues may be at least about 7 amino acid residues apart.The two amino acid residues may be at least about 11 amino acid residuesapart. The two amino acid residues may be at least about 15 amino acidresidues apart. The two amino acid residues may be at least about 19amino acid residues apart.

The staple may be conjugated to one or more lipids to produce astaple-lipid construct. Exemplary staple lipid constructs are shown inFIG. 3A-H. FIG. 3A-C depict a staple lipid construct comprising (a) astaple (C); and (b) a lipid (L). The lipid may be attached to anyportion of the staple. As shown in FIG. 3A, the lipid (L) is attached toan internal region of the staple (C). As shown in FIGS. 3B and 3C, thelipid (L) is attached to one end of the staple (C). FIG. 3D-F depict astaple lipid construct comprising (a) a staple (C); and (b) two lipids(L¹ and L²). As shown in FIG. 3D, the first lipid (L¹) is attached toone end of the staple (C) and the second lipid (L²) is attached to aninternal region of the staple (C). As shown in FIG. 3E, the first lipid(L¹) and the second lipid (L²) are attached opposite ends of the staple(C). As shown in FIG. 3F, the first lipid (L¹) is attached to aninternal region of the staple (C) and the second lipid (L²) is attachedto one end of the staple (C). The staple lipid construct may comprise(a) a staple (C); and (b) a plurality of lipids (L¹ . . . L^(n)). FIG.3G depicts a staple lipid construct comprising (a) a staple (C); and (b)three lipids (L¹, L² and L³). As shown in FIG. 3G, the three lipids (L¹,L² and L³) are attached to various regions within the staple (C). FIG.3H depicts a staple lipid construct comprising (a) a staple (C); and (b)four lipids (L¹, L², L² and L³). As shown in FIG. 3G, the four lipids(L¹, L², L² and L³) are attached to various regions within the staple(C). The staple lipid constructs may further comprise one or moreadditional staples. The one or more additional staples may be attachedto the staple, lipid, or a combination thereof. For example, the one ormore additional staples may be attached to the staple and the lipid.Alternatively, the one or more additional staples are attached to thestaple. The one or more additional staples may be attached to two ormore staples. The one or more additional staples may be attached to thelipid. The one or more additional staples may be attached to the two ormore lipids.

The staple may be prepared from a precursor compound comprising two ormore chemical groups. The two or more chemical groups may react with anucleophilic or electrophilic amino acid residue on the same TA. The twoor more chemical groups may react with a nucleophilic amino acid residueon the same TA. The two or more chemical groups may react with anelectrophilic amino acid residue on the same TA. The two or morechemical groups may react with a nucleophilic or electrophilic aminoacid residue on two or more TAs. The two or more chemical groups mayreact with a nucleophilic amino acid residue on two or more TAs. The twoor more chemical groups may react with an electrophilic amino acidresidue on two or more TAs.

The staple may be prepared from a precursor compound with two chemicalgroups, each of which reacts with a nucleophilic or electrophilic aminoacid residue on one or more TAs. The staple may be prepared from aprecursor compound with two chemical groups, each of which reacts with anucleophilic amino acid residue on the same TA. The staple may beprepared from a precursor compound with two chemical groups, each ofwhich reacts with an electrophilic amino acid residue on the same TA.

The precursor compound may further comprise one or more additionalchemical groups that react with one or more lipid derivatives to formthe PLC or mTA. Additional chemical groups include, but are not limitedto, alkenes, alkynes, amines, hydrazines, azides, carboxylic acids,esters, anilines, ketones, ketoamides, and hydroxy groups.

The precursor compound may be selected from:

wherein

-   -   each W is independently selected from Cl, Br, I, and maleimide;    -   each X is independently selected from O, NH, and S;    -   each Y is independently selected from N and CH;    -   each Z is independently selected from N and CH;    -   a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or        17;    -   b is 1, 2, 3, 4, 5, or 6; and    -   c is 1 or 2.

The precursor compound may be selected from:

wherein

-   -   each W is independently selected from Cl, Br, I, and maleimide;    -   X is O, NH, or S; and    -   Y is N or CH.

The precursor may be selected from Table 2.

At least one or more lipids of the PLC of Formula (II), Formula (III),or Formula (IV) may be attached to the one or more staples to form alipid staple precursor prior to forming the peptide conjugate. Each ofthe lipids of the PLC of Formula (II), Formula (III), or Formula (IV)may be attached to the one or more staples to form a lipid stapleprecursor prior to forming the peptide conjugate. The lipid stapleprecursor may be

wherein each W is independently selected from Cl, Br, I, and maleimide;c is 1 or 2; and L is the lipid.Therapeutic Agent (TA)

The mTAs disclosed herein may comprise one or more therapeutic agents.The mTAs may comprise one therapeutic agent. The mTAs may comprise twotherapeutic agents. The mTAs may comprise 3, 4, 5, 6, 7 or moretherapeutic agents. The therapeutic agents may be different. Thetherapeutic agents may be the same.

The PCs disclosed herein may comprise one or more therapeutic agents.The PCs may comprise two or more therapeutic agents. The PCs maycomprise 3, 4, 5, 6, 7 or more therapeutic agents. The two or moretherapeutic agents may be different. The two or more therapeutic agentsmay be the same. Exemplary TAs are depicted in Tables 3, 4, and 5.Exemplary TAs may comprise a peptide sequence disclosed in Tables 3, 4,and 5. Exemplary TAs may comprise a polynucleotide encoding a peptidedisclosed in Tables 3, 4, and 5.

The TA may be selected from peptides listed Table 5, wherein X iscysteine. The TA may be selected from analogs of peptides listed inTable 5, wherein X is homocysteine.

In some embodiments, the TA may be a modified therapeutic peptide with aD-serine in place of L-serine. In some embodiments, the TA may be amodified therapeutic peptide with an aminoisobutyric acid (Aib) in placeof L-serine.

The TA may be a hormone. Examples of hormones include, but are notlimited to, peptide hormones, lipid and phospholipid-derived hormones,and monoamines. Peptide hormones generally consist of chains of aminoacids. Examples of small peptide hormones include, but are not limitedto thyrotropin-releasing hormone (TRH) and vasopressin. Peptidescomposed of scores or hundreds of amino acids are referred to asproteins. Examples of protein hormones include insulin and growthhormone. More complex protein hormones may bear carbohydrate side-chainsand may be called glycoprotein hormones. Luteinizing hormone,follicle-stimulating hormone and thyroid-stimulating hormone areexamples of glycoprotein hormones. Lipid and phospholipid-derivedhormones are generally derived from lipids such as linoleic acid andarachidonic acid and phospholipids. Examples of protein hormones maycomprise steroid hormones that may be derived from cholesterol and theeicosanoids. Examples of steroid hormones are testosterone and cortisol.Eicosanoids may comprise prostaglandins. Monoamines may be derived fromaromatic amino acids like phenylalanine, tyrosine, and tryptophan by theaction of aromatic amino acid decarboxylase enzymes. The TA may beleptin. The TA may be betatrophin. The TA may be a peptide agonist orpeptide hormone. The peptide agonist or hormone may be exendin-4,exenatide, glucagon, glucagon-like protein-1 (GLP-1), or oxyntomodulin.The TA may be exendin-4. The TA may be exenatide. The TA may beglucagon. The TA may be glucagon-like protein-1 (GLP-1). The TA may beoxyntomodulin. The TA may be GLP-2. The TA may be a GLP-1R and GIPR dualagonist. The TA may be a GLP-1R and GCGR dual agonist. The TA may be aGLP1R, GCGR and GIPR tri-agonist. The TA may bind to a receptor. Thereceptor may be a GLP-1 receptor or glucagon receptor. The TA may beselected from GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, GIP,GLP1R and GCGR dual agonist, GLP1R and GIPR dual agonist, and GLP1R,GCGR and GIPR tri-agonist. The TA may be selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP.

The TA may be a growth factor. Growth factors may include, but are notlimited to, cytokines and hormones. Examples of growth factors include,but are not limited to, adrenomedullin (AM), angiopoietin (Ang),autocrine motility factor, bone morphogenetic proteins (BMPs),brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF),erythropoietin (EPO), fibroblast growth factor (FGF), glial cellline-derived neurotrophic factor (GDNF), granulocyte colony-stimulatingfactor (G-CSF), granulocyte macrophage colony-stimulating factor(GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growthfactor (HGF), hepatoma-derived growth factor (HDGF), insulin-like growthfactor (IGF), migration-stimulating factor, myostatin (GDF-8), nervegrowth factor (NGF) and other neurotrophins, platelet-derived growthfactor (PDGF), thrombopoietin (TPO), transforming growth factoralpha(TGF-α), transforming growth factor beta(TGF-β), tumor necrosisfactor-alpha(TNF-α) and vascular endothelial growth factor (VEGF). TheTA may be fibroblast growth factor 21 (FGF21).

The TA may be a cell regulatory protein. The TA may be a cell regulatoryprotein of the transforming growth factor beta superfamily. The TA maybe a member of the decapentaplegic-Vg related (DVR) related subfamily.The TA may be a member of the activin/inhibin subfamily. The TA may be amember of the TGF-beta subfamily. The TA may be a growth differentiationfactor (GDF). The GDF may be GDF1, GDF2, GDF3, GDF5, GDF6, GFD8, GDF9,GDF10, GDF11, and GDF15. The TA may be growth differentiation factor 11(GDF11).

The TA may be a protein. The protein may be a member of theangiopoietin-like family of secreted factors. The protein may be anangiopoietin-like protein (ANGPTL). Examples of ANGPTLs include, but arenot limited to, ANGPTL1, ANGPTL2, ANGPTL3, ANGPTL4, ANGPTL5, ANGPTL6 andANGPTL7. The TA may be ANGPTL3.

The TA may comprise an amino acid sequence selected from the groupcomprising SEQ ID NO: 1-6. The TA may comprise an amino acid sequencethat is at least about 50% homologous to an amino acid sequence selectedfrom the group comprising SEQ ID NO: 1-6. The TA may comprise an aminoacid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 97%, 99%, or 100% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 1-6. The TA may comprisean amino acid sequence that is at least about 70% homologous to an aminoacid sequence selected from the group comprising SEQ ID NO: 1-6. The TAmay comprise an amino acid sequence that is at least about 75%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 1-6. The TA may comprise an amino acid sequence that is atleast about 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 1-6.

The TA may comprise 20 or more consecutive amino acids from an aminoacid sequence selected from the group comprising SEQ ID NO: 1-6. The TAmay comprise 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130 or moreconsecutive amino acids from an amino acid sequence selected from thegroup comprising SEQ ID NO: 1-6.

The TA may comprise an amino acid sequence selected from the groupcomprising SEQ ID NO: 7-53. The TA may comprise an amino acid sequencethat is at least about 50% homologous to an amino acid sequence selectedfrom the group comprising SEQ ID NO: 7-53. The TA may comprise an aminoacid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 97%, 99%, or 100% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 7-53. The TA may comprisean amino acid sequence that is at least about 70% homologous to an aminoacid sequence selected from the group comprising SEQ ID NO: 7-53. The TAmay comprise an amino acid sequence that is at least about 75%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 7-53. The TA may comprise an amino acid sequence that is atleast about 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 7-53.

The TA may comprise 10 or more consecutive amino acids from an aminoacid sequence selected from the group comprising SEQ ID NO: 7-53. The TAmay comprise 5, 6, 7, 8, 9, 10 or more consecutive amino acids from anamino acid sequence selected from the group comprising SEQ ID NO: 7-53.

The TA may comprise an amino acid sequence selected from the groupcomprising SEQ ID NO: 8-12. The TA may comprise an amino acid sequencethat is at least about 50% homologous to an amino acid sequence selectedfrom the group comprising SEQ ID NO: 8-12. The TA may comprise an aminoacid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 97%, 99%, or 100% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 8-12. The TA may comprisean amino acid sequence that is at least about 70% homologous to an aminoacid sequence selected from the group comprising SEQ ID NO: 8-12. The TAmay comprise an amino acid sequence that is at least about 75%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 8-12. The TA may comprise an amino acid sequence that is atleast about 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 8-12.

The TA may comprise 10 or more consecutive amino acids from an aminoacid sequence selected from the group comprising SEQ ID NO: 8-12. The TAmay comprise 5, 6, 7, 8, 9, 10 or more consecutive amino acids from anamino acid sequence selected from the group comprising SEQ ID NO: 8-12.

The TA may comprise an amino acid sequence selected from the groupcomprising SEQ ID NO: 15-19. The TA may comprise an amino acid sequencethat is at least about 50% homologous to an amino acid sequence selectedfrom the group comprising SEQ ID NO: 15-19. The TA may comprise an aminoacid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 97%, 99%, or 100% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 15-19. The TA may comprisean amino acid sequence that is at least about 70% homologous to an aminoacid sequence selected from the group comprising SEQ ID NO: 15-19. TheTA may comprise an amino acid sequence that is at least about 75%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 15-19. The TA may comprise an amino acid sequence that is atleast about 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 15-19.

The TA may comprise 10 or more consecutive amino acids from an aminoacid sequence selected from the group comprising SEQ ID NO: 15-19. TheTA may comprise 5, 6, 7, 8, 9, 10 or more consecutive amino acids froman amino acid sequence selected from the group comprising SEQ ID NO:15-19.

The TA may comprise an amino acid sequence selected from the groupcomprising SEQ ID NO: 20-29. The TA may comprise an amino acid sequencethat is at least about 50% homologous to an amino acid sequence selectedfrom the group comprising SEQ ID NO: 20-29. The TA may comprise an aminoacid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 97%, 99%, or 100% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 20-29. The TA may comprisean amino acid sequence that is at least about 70% homologous to an aminoacid sequence selected from the group comprising SEQ ID NO: 20-29. TheTA may comprise an amino acid sequence that is at least about 75%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 20-29. The TA may comprise an amino acid sequence that is atleast about 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 20-29.

The TA may comprise 10 or more consecutive amino acids from an aminoacid sequence selected from the group comprising SEQ ID NO: 20-29. TheTA may comprise 5, 6, 7, 8, 9, 10 or more consecutive amino acids froman amino acid sequence selected from the group comprising SEQ ID NO:20-29.

The TA may comprise an amino acid sequence selected from the groupcomprising SEQ ID NO: 30-53. The TA may comprise an amino acid sequencethat is at least about 50% homologous to an amino acid sequence selectedfrom the group comprising SEQ ID NO: 30-53. The TA may comprise an aminoacid sequence that is at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 92%, 95%, 97%, 99%, or 100% homologous to an amino acid sequenceselected from the group comprising SEQ ID NO: 30-53. The TA may comprisean amino acid sequence that is at least about 70% homologous to an aminoacid sequence selected from the group comprising SEQ ID NO: 30-53. TheTA may comprise an amino acid sequence that is at least about 75%homologous to an amino acid sequence selected from the group comprisingSEQ ID NO: 30-53. The TA may comprise an amino acid sequence that is atleast about 80% homologous to an amino acid sequence selected from thegroup comprising SEQ ID NO: 30-53.

The TA may comprise 10 or more consecutive amino acids from an aminoacid sequence selected from the group comprising SEQ ID NO: 30-53. TheTA may comprise 5, 6, 7, 8, 9, 10 or more consecutive amino acids froman amino acid sequence selected from the group comprising SEQ ID NO:30-53.

The TA may comprise one or more cysteine residues. The one or morecysteine residues may be used for connecting the staple to the TA. Theone or more cysteine residues may be used for connecting the lipid tothe TA. The one or more cysteine residues may be used for connecting afirst TA to a second TA. The one or more cysteine residues may benaturally occurring in the TA. Alternatively, the one or more cysteineresidues are introduced into the TA. The one or more cysteine residuesmay be inserted into the TA. The one or more amino acid residues mayreplace one or more amino acid residues in the TA. Methods for aminoacid substitution and/or insertion are known in the art.

The one or more TAs may comprise a polypeptide derivative. Thepolypeptide derivative may comprise at least a portion of a wild-typepolypeptide comprising one or more amino acid mutations. The one or moreamino acid mutations may comprise a deletion, substitution, addition ora combination thereof. The one or more amino acid mutations may compriseadding one or more amino acid residues to a wild-type polypeptide. Theone or more amino acid mutations may comprise deletion of one or moreamino acid residues of the wild-type polypeptide. The one or more aminoacid mutations may comprise substitution of one or more amino acidresidues of the wild-type polypeptide. The one or more amino acidmutations may comprise substituting one or more amino acid residues ofthe wild-type polypeptide with one or more cysteine residues. The one ormore amino acid mutations may comprise substituting one or more aminoacid residues of the wild-type polypeptide with one or more D-amino acidresidues. The one or more amino acid residues of the wild-typepolypeptide may comprise one or more alanines, methionines, arginines,serines, threonines, and tyrosines.

The one or more TAs may comprise a modified therapeutic peptide. Methodsof modifying peptides are known in the art (see for example, GentilucciL et al., 2010, Curr Pharm Des). Examples of peptide modificationsinclude, but are not limited to, acetylation, phosphorylation, andmethylation. The peptide modification may comprise a chemicalmodification. Peptide modifications may occur on the N-terminus of thepeptide. Alternatively, or additionally, peptide modifications may occuron the C-terminus of the peptide. Peptide modifications may occur at oneor more internal amino acids of the peptide. Peptide modifications maycomprise replacing the carboxyl group at the C-terminus of the peptide.Peptide modifications may comprise modifying the carboxyl group at theC-terminus of the peptide. The carboxyl group at the C-terminus of thepeptide may be modified to produce an amide group. The carboxyl group atthe C-terminus of the peptide may be modified to produce an amine group.

The one or more staples of the PLC or mTA may be attached to two or moreresidues in the one or more TAs. The TA may comprise a fusion peptide.The two or more residues may be adjacent. The two or more residues maybe non-adjacent. The two or more residues may be at least about 1 aminoacid residue apart. The two or more residues may be at least about 2, 3,4, 5 or more amino acid residues apart. The two or more residues may beat least about 4 amino acid residues apart. The two or more residues maybe at least about 5 amino acid residues apart. The two or more residuesmay be at least about 6 amino acid residues apart. The two or moreresidues may be at least about 7 amino acid residues apart. The two ormore residues may be at least about 8 amino acid residues apart. The twoor more residues may be at least about 9 amino acid residues apart. Thetwo or more residues may be at least about 10 amino acid residues apart.The two or more residues may be at least about 11 amino acid residuesapart. The two or more residues may be at least about 12 amino acidresidues apart. The two or more residues may be at least about 13 aminoacid residues apart. The two or more residues may be at least about 14amino acid residues apart. The two or more residues may be at leastabout 15 amino acid residues apart. The two or more residues may be atleast about 4, 7, 11, or 14 amino acid residues apart. The two or moreresidues may be less than about 100 amino acid residues apart. The twoor more residues may be less than about 90, 85, 80, 75, 70, 65, 60, 55amino acid residues apart. The two or more residues may be less thanabout 50 amino acid residues apart. The two or more residues may be lessthan about 30 amino acid residues apart. The two or more residues may beless than about 20 amino acid residues apart.

The TAs may be from a mammal or non-mammal. The TAs may be from a human.Alternatively, the TAs may be from a goat, sheep, cow, rabbit, monkey,dog, cat or a combination thereof. The TAs may be from a reptile. TheTAs may be from a snake or lizard. The TAs may be from an amphibian. TheTAs may be from a frog or toad. The TAs may be from an avian. The TAsmay be recombinant peptide.

Linker

The PLCs or mTAs disclosed herein may further comprise one or morelinkers. The PLCs or mTAs disclosed herein may further comprise two ormore linkers. The PLCs or mTAs disclosed herein may further comprisethree or more linkers. The PLCs or mTAs disclosed herein may furthercomprise four or more linkers. The PLCs or mTAs disclosed herein mayfurther comprise five or more linkers.

The one or more linkers may enable attachment of a lipid to a peptideconjugate. The one or more linkers may enable attachment of a lipid to atherapeutic agent. The one or more linkers may enable attachment of alipid to a staple. The linker may enable attachment of a lipid toanother lipid. The linker may enable attachment of a lipid to a chemicalgroup comprising one or more polyethylene glycol subunits. The linkermay enable attachment of a PEG to another PEG. The linker may enableattachment of a PEG to a therapeutic agent. The linker may enableattachment of a therapeutic agent to another therapeutic agent. Thelinker may be an amino acid. The linker may be an amino acid of thetherapeutic agent. The linker may be a substituted amino acid of thetherapeutic agent. The linker may be a cysteine. The linker may be anL-cysteine. The linker may be an ether or an amide. The linker may be athioether. The linker may be a carbamate. The linker may be a Michaelreaction adduct. The linker may link a PEG molecule to a lipid.

The linker may be formed by reaction of an amino acid on the peptideregion with an electrophilic linker precursor. The linker may be formedby reaction of a cysteine on the peptide region with an electrophiliclinker precursor. The electrophilic linker precursor may be a stapleprecursor compound, a lipid staple precursor, or a lipid derivative. Thelinker may be formed by reaction of a derivatizable functional group onthe staple precursor compound with a lipid derivative to produce a lipidstaple precursor. The linker may be formed by reaction of aderivatizable functional group on the staple already attached to thetherapeutic agent with a lipid derivative.

The linker may be the product of a bioorthogonal reaction. The linkermay be an oxime, a tetrazole, a Diels Alder adduct, a hetero Diels Alderadduct, an aromatic substitution reaction product, a nucleophilicsubstitution reaction product, an ester, an amide, a carbamate, or aMichael reaction product. The linker may be a metathesis reactionproduct, a metal-mediated cross-coupling reaction product, a radicalpolymerization product, an oxidative coupling product, an acyl-transferreaction product, or a photo click reaction product.

Pharmacokinetics

Mechanisms by which the PLCs or mTAs positively influencepharmacokinetic or pharmacodynamic behavior include, but are not limitedto, (i) preventing or mitigating in vivo proteolytic degradation orother activity-diminishing chemical modification of the therapeuticagent; (ii) improving half-life or other pharmacokinetic properties byreducing renal filtration, decreasing receptor-mediated clearance orincreasing bioavailability; (iii) reducing toxicity; (iv) improvingsolubility; and/or (v) increasing biological activity and/or targetselectivity of the therapeutic agent or unmodified therapeutic peptide.

The PLCs or mTAs may enhance one or more pharmacokinetic properties of atherapeutic agent (TA) when attached to the TA. The PLCs or mTAsdisclosed herein may enhance the one or more pharmacokinetic propertiesof the TA by at least about 200% as measured by pharmacodynamics whencompared to the TA or unmodified therapeutic peptide alone. The PLCs ormTAs disclosed herein may enhance the one or more pharmacokineticproperties of the TA by at least about 300%, 400%, 500%, 600%, 700%,800%, 900%, 1000% as measured by pharmacodynamics when compared to theTA or unmodified therapeutic peptide alone.

The pharmacokinetic properties may comprise a half-life. The half-lifeof the PLC or mTA may be at least about two-fold longer compared to thehalf-life of the TA or unmodified therapeutic peptide alone. Thehalf-life of the PLC or mTA disclosed herein may be at least about3-fold, 4-fold, or 5-fold longer compared to the half-life of the TA orunmodified therapeutic peptide alone. The half-life of the PLC or mTAdisclosed herein may be at least about 6-, 7-, 8-, 9-, 10-, 15-, 20-,25-, 30-, 35-, 40-, 45-, or 50-fold longer compared to the half-life ofthe TA or unmodified therapeutic peptide alone.

In addition, the PLCs or mTAs may have positive effects on terms ofincreasing manufacturability, and/or reducing immunogenicity of thetherapeutic agent, compared to an unconjugated form of the therapeuticagent or unmodified therapeutic peptide.

Therapeutic Use

Further disclosed herein are PLCs or mTAs for treating, alleviating,inhibiting and/or preventing one or more diseases and/or conditions. Thedisease and/or condition may be a chronic disease or condition.Alternatively, the disease and/or condition is an acute disease orcondition. The disease or condition may be recurrent, refractory,accelerated, or in remission. The disease or condition may affect one ormore cell types. The one or more diseases and/or conditions may be anautoimmune disease, inflammatory disease, or metabolic disease.

Disclosed herein are methods for treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids; and (b) one or morepeptide conjugates (PC), the peptide conjugate (PC) comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more peptide conjugates. The oneor more lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. The two or moreresidues in the peptide region may comprise cysteine. The one or moreTAs may comprise a GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2,GIP, GLP1R and GCGR dual agonist, GLP1R and GIPR dual agonist, or GLP1R,GCGR and GIPR tri-agonist, or a derivative thereof. The disease orcondition may be a diabetes or obesity, or a medical conditionassociated with diabetes or obesity. The disease or condition may benon-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis(NASH), or cardiovascular disease. The disease or condition may be anautoimmune disorder. The disease or condition may be Crohn's disease orulcerative colitis. The disease or condition may be short bowel syndrome(SBS). The disease or condition may be inflammatory bowel disease (IBD),inflammatory bowel syndrome (IBS), or psoriasis. The disease orcondition may be Alzheimer's disease, Parkinson's disease orHuntington's disease. The PLC may be administered with one or moreadditional therapeutic agents. The additional therapeutic agents maycomprise one or more other diabetes drugs, DPP4 inhibitors, SGLT2inhibitors, hypoglycemic drugs and biguanidine drugs, insulinsecretogogues and sulfonyl urea drugs, TZD drugs, insulin and insulinanalogs, FGF21 and analogs, leptin or leptin analogs, amylin and amylinanalogs, an anti-inflammatory drug, cyclosporine A or FK506, 5-ASA, or astatin, or any combination thereof. The additional therapeutic agent maybe aspirin.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids, the lipids selectedfrom a group consisting of sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols; and (b) one or more peptide conjugates (PC), the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, wherein the one or more lipids are attached to the oneor more peptide conjugates. The one or more lipids may be attached tothe one or more TAs. The one or more lipids may be attached to the oneor more lipids. The two or more residues in the peptide region maycomprise cysteine. The one or more TAs may comprise GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, GIP, GLP1R and GCGR dual agonist, GLP1Rand GIPR dual agonist, or GLP1R, GCGR and GIPR tri-agonist, or aderivative thereof. The disease or condition may be a diabetes orobesity, or a medical condition associated with diabetes or obesity. Thedisease or condition may be non-alcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), or cardiovascular disease. Thedisease or condition may be an autoimmune disorder. The disease orcondition may be Crohn's disease or ulcerative colitis. The disease orcondition may be short bowel syndrome (SBS). The disease or conditionmay be inflammatory bowel disease (IBD), inflammatory bowel syndrome (BS), or psoriasis. The disease or condition may be Alzheimer's disease,Parkinson's disease or Huntington's disease. The PLC may be administeredwith one or more additional therapeutic agents. The additionaltherapeutic agents may comprise one or more other diabetes drugs, DPP4inhibitors, SGLT2 inhibitors, hypoglycemic drugs and biguanidine drugs,insulin secretogogues and sulfonyl urea drugs, TZD drugs, insulin andinsulin analogs, FGF21 and analogs, leptin or leptin analogs, amylin andamylin analogs, an anti-inflammatory drug, cyclosporine A or FK506,5-ASA, or a statin, or any combination thereof. The additionaltherapeutic agent may be aspirin. The additional therapeutic agent maybe aspirin.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more peptide lipid conjugates (PLCs) of Formula (II):PC-A¹-P¹-L, wherein PC is a peptide conjugate comprising (a) one or morepeptide regions comprising one or more peptide therapeutic agents (TAs);and (b) one or more staples, wherein the staples connect two or moreresidues in the peptide region; A¹ is a chemical group linking PC andP¹; P¹ is a bond or -PEG-A²-; PEG is a chemical group comprising one ormore polyethylene glycol subunits; A² is a chemical group linking PEGand L; and L is a lipid. The two or more residues in the peptide regionmay comprise cysteine. The one or more TAs may comprise GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, GIP, GLP1R and GCGR dual agonist, GLP1Rand GIPR dual agonist, or GLP1R, GCGR and GIPR tri-agonist, or aderivative thereof. The disease or condition may be a diabetes orobesity, or a medical condition associated with diabetes or obesity. Thedisease or condition may be non-alcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), or cardiovascular disease. Thedisease or condition may be an autoimmune disorder. The disease orcondition may be Crohn's disease or ulcerative colitis. The disease orcondition may be short bowel syndrome (SBS). The disease or conditionmay be inflammatory bowel disease (IBD), inflammatory bowel syndrome(IBS), or psoriasis. The disease or condition may be Alzheimer'sdisease, Parkinson's disease or Huntington's disease. The PLC may beadministered with one or more additional therapeutic agents. Theadditional therapeutic agents may comprise one or more other diabetesdrugs, DPP4 inhibitors, SGLT2 inhibitors, hypoglycemic drugs andbiguanidine drugs, insulin secretogogues and sulfonyl urea drugs, TZDdrugs, insulin and insulin analogs, FGF21 and analogs, leptin or leptinanalogs, amylin and amylin analogs, an anti-inflammatory drug,cyclosporine A or FK506, 5-ASA, or a statin, or any combination thereof.The additional therapeutic agent may be aspirin.

Disclosed herein are methods for treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids; and (b) one or morepeptide conjugates (PC), the peptide conjugate (PC) comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more staples. At least one of thetwo or more residues may comprise a cysteine. The two or more residuesin the peptide region may comprise cysteine. In some instances, at leastone of the two or more residues is not cysteine. In some instances, atleast two of the two or more residues are not cysteine. The one or moreTAs may comprise a GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2,GIP, GLP1R and GCGR dual agonist, GLP1R and GIPR dual agonist, or GLP1R,GCGR and GIPR tri-agonist, or a derivative thereof. The disease orcondition may be a diabetes or obesity, or a medical conditionassociated with diabetes or obesity The disease or condition may benon-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis(NASH), or cardiovascular disease. The disease or condition may be anautoimmune disorder. The disease or condition may be Crohn's disease orulcerative colitis. The disease or condition may be short bowel syndrome(SBS). The disease or condition may be inflammatory bowel disease (IBD),inflammatory bowel syndrome (IBS), or psoriasis. The disease orcondition may be Alzheimer's disease, Parkinson's disease orHuntington's disease. The PLC may be administered with one or moreadditional therapeutic agents. The additional therapeutic agents maycomprise one or more other diabetes drugs, DPP4 inhibitors, SGLT2inhibitors, hypoglycemic drugs and biguanidine drugs, insulinsecretogogues and sulfonyl urea drugs, TZD drugs, insulin and insulinanalogs, FGF21 and analogs, leptin or leptin analogs, amylin and amylinanalogs, an anti-inflammatory drug, cyclosporine A or FK506, 5-ASA, or astatin, or any combination thereof. The additional therapeutic agent maybe aspirin.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids, the lipids selectedfrom a group consisting of sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols; and (b) one or more peptide conjugates (PC), the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, wherein the one or more lipids are attached to the oneor more staples. At least one of the two or more residues may comprise acysteine. The two or more residues in the peptide region may comprisecysteine. In some instances, at least one of the two or more residues isnot cysteine. In some instances, at least two of the two or moreresidues are not cysteine. The one or more TAs may comprise a GLP-1,glucagon, oxyntomodulin, exendin-4, GLP-2, GIP, GLP1R and GCGR dualagonist, GLP1R and GIPR dual agonist, or GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof. The disease or condition may be adiabetes or obesity, or a medical condition associated with diabetes orobesity. The disease or condition may be non-alcoholic fatty liverdisease (NAFLD), nonalcoholic steatohepatitis (NASH), or cardiovasculardisease. The disease or condition may be an autoimmune disorder. Thedisease or condition may be Crohn's disease or ulcerative colitis. Thedisease or condition may be short bowel syndrome (SBS). The disease orcondition may be inflammatory bowel disease (IBD), inflammatory bowelsyndrome (B S), or psoriasis. The disease or condition may beAlzheimer's disease, Parkinson's disease or Huntington's disease. ThePLC may be administered with one or more additional therapeutic agents.The additional therapeutic agents may comprise one or more otherdiabetes drugs, DPP4 inhibitors, SGLT2 inhibitors, hypoglycemic drugsand biguanidine drugs, insulin secretogogues and sulfonyl urea drugs,TZD drugs, insulin and insulin analogs, FGF21 and analogs, leptin orleptin analogs, amylin and amylin analogs, an anti-inflammatory drug,cyclosporine A or FK506, 5-ASA, or a statin, or any combination thereof.The additional therapeutic agent may be aspirin.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more peptide lipid conjugates (PLCs) of Formula (III). Atleast one of the two or more residues may comprise a cysteine. The twoor more residues in the peptide region may comprise cysteine. In someinstances, at least one of the two or more residues is not cysteine. Insome instances, at least two of the two or more residues are notcysteine. The one or more TAs may comprise a GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, GIP, GLP1R and GCGR dual agonist, GLP1Rand GIPR dual agonist, or GLP1R, GCGR and GIPR tri-agonist, or aderivative thereof. The disease or condition may be a diabetes orobesity, or a medical condition associated with diabetes or obesity. Thedisease or condition may be non-alcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), or cardiovascular disease. Thedisease or condition may be an autoimmune disorder. The disease orcondition may be Crohn's disease or ulcerative colitis. The disease orcondition may be short bowel syndrome (SBS). The disease or conditionmay be inflammatory bowel disease (IBD), inflammatory bowel syndrome (BS), or psoriasis. The disease or condition may be Alzheimer's disease,Parkinson's disease or Huntington's disease. The PLC may be administeredwith one or more additional therapeutic agents. The additionaltherapeutic agents may comprise one or more other diabetes drugs, DPP4inhibitors, SGLT2 inhibitors, hypoglycemic drugs and biguanidine drugs,insulin secretogogues and sulfonyl urea drugs, TZD drugs, insulin andinsulin analogs, FGF21 and analogs, leptin or leptin analogs, amylin andamylin analogs, an anti-inflammatory drug, cyclosporine A or FK506,5-ASA, or a statin, or any combination thereof. The additionaltherapeutic agent may be aspirin.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more peptide lipid conjugates (PLCs) of Formula (IV). Atleast one of the two or more residues may comprise a cysteine. The twoor more residues in the peptide region may comprise cysteine. In someinstances, at least one of the two or more residues is not cysteine. Insome instances, at least two of the two or more residues are notcysteine. The one or more TAs may comprise a GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, GIP, GLP1R and GCGR dual agonist, GLP1Rand GIPR dual agonist, or GLP1R, GCGR and GIPR tri-agonist, or aderivative thereof. The disease or condition may be a diabetes orobesity, or a medical condition associated with diabetes or obesity. Thedisease or condition may be non-alcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), or cardiovascular disease. Thedisease or condition may be an autoimmune disorder. The disease orcondition may be Crohn's disease or ulcerative colitis. The disease orcondition may be short bowel syndrome (SBS). The disease or conditionmay be inflammatory bowel disease (IBD), inflammatory bowel syndrome (BS), or psoriasis. The disease or condition may be Alzheimer's disease,Parkinson's disease or Huntington's disease. The PLC may be administeredwith one or more additional therapeutic agents. The additionaltherapeutic agents may comprise one or more other diabetes drugs, DPP4inhibitors, SGLT2 inhibitors, hypoglycemic drugs and biguanidine drugs,insulin secretogogues and sulfonyl urea drugs, TZD drugs, insulin andinsulin analogs, FGF21 and analogs, leptin or leptin analogs, amylin andamylin analogs, an anti-inflammatory drug, cyclosporine A or FK506,5-ASA, or a statin, or any combination thereof. The additionaltherapeutic agent may be aspirin.

Disclosed herein are methods for treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) two or more lipids; and (b) one or morepeptide conjugates (PC), the peptide conjugate (PC) comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein at least oneof the two or more lipids are attached to the one or more therapeuticagents and at least one of the two or more lipids are attached to theone or more staples. At least one of the two or more residues maycomprise a cysteine. The two or more residues in the peptide region maycomprise cysteine. In some instances, at least one of the two or moreresidues is not cysteine. In some instances, at least two of the two ormore residues are not cysteine. The one or more TAs may comprise aGLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, GIP, GLP1R and GCGRdual agonist, GLP1R and GIPR dual agonist, or GLP1R, GCGR and GIPRtri-agonist, or a derivative thereof. The disease or condition may be adiabetes or obesity, or a medical condition associated with diabetes orobesity. The disease or condition may be non-alcoholic fatty liverdisease (NAFLD), nonalcoholic steatohepatitis (NASH), or cardiovasculardisease. The disease or condition may be an autoimmune disorder. Thedisease or condition may be Crohn's disease or ulcerative colitis. Thedisease or condition may be short bowel syndrome (SBS). The disease orcondition may be inflammatory bowel disease (IBD), inflammatory bowelsyndrome (IBS), or psoriasis. The disease or condition may beAlzheimer's disease, Parkinson's disease or Huntington's disease. ThePLC may be administered with one or more additional therapeutic agents.The additional therapeutic agents may comprise one or more otherdiabetes drugs, DPP4 inhibitors, SGLT2 inhibitors, hypoglycemic drugsand biguanidine drugs, insulin secretogogues and sulfonyl urea drugs,TZD drugs, insulin and insulin analogs, FGF21 and analogs, leptin orleptin analogs, amylin and amylin analogs, an anti-inflammatory drug,cyclosporine A or FK506, 5-ASA, or a statin, or any combination thereof.The additional therapeutic agent may be aspirin.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) two or more lipids, the lipids selectedfrom a group consisting of sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols; and (b) one or more peptide conjugates (PC), the peptideconjugate comprising a peptide region comprising one or more peptidetherapeutic agents (TA) and a staple region comprising one or morestaples, the one or more staples connect two or more residues in thepeptide region, wherein at least one of the two or more lipids areattached to the one or more therapeutic agents and at least one of thetwo or more lipids are attached to the one or more staples. At least oneof the two or more residues may comprise a cysteine. The two or moreresidues in the peptide region may comprise cysteine. In some instances,at least one of the two or more residues is not cysteine. In someinstances, at least two of the two or more residues are not cysteine.The one or more TAs may comprise a GLP-1, glucagon, oxyntomodulin,exendin-4, GLP-2, GIP, GLP1R and GCGR dual agonist, GLP1R and GIPR dualagonist, or GLP1R, GCGR and GIPR tri-agonist, or a derivative thereof.The disease or condition may be a diabetes or obesity, or a medicalcondition associated with diabetes or obesity. The disease or conditionmay be non-alcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), or cardiovascular disease. The disease orcondition may be an autoimmune disorder. The disease or condition may beCrohn's disease or ulcerative colitis. The disease or condition may beshort bowel syndrome (SBS). The disease or condition may be inflammatorybowel disease (IBD), inflammatory bowel syndrome (IBS), or psoriasis.The disease or condition may be Alzheimer's disease, Parkinson's diseaseor Huntington's disease. The PLC may be administered with one or moreadditional therapeutic agents. The additional therapeutic agents maycomprise one or more other diabetes drugs, DPP4 inhibitors, SGLT2inhibitors, hypoglycemic drugs and biguanidine drugs, insulinsecretogogues and sulfonyl urea drugs, TZD drugs, insulin and insulinanalogs, FGF21 and analogs, leptin or leptin analogs, amylin and amylinanalogs, an anti-inflammatory drug, cyclosporine A or FK506, 5-ASA, or astatin, or any combination thereof. The additional therapeutic agent maybe aspirin.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more PLCs, wherein the one or morepeptide lipid conjugates (PLCs) comprise (a) one or more lipids; and (b)one or more peptide conjugates (PC), the peptide conjugate (PC)comprising a peptide region comprising one or more peptide therapeuticagents (TA) and a staple region comprising one or more staples, the oneor more staples connect two or more residues in the peptide region,wherein the one or more lipids are attached to the one or more peptideconjugates. The one or more lipids may be attached to the one or moreTAs. The one or more lipids may be attached to the one or more staples.At least one of the two or more residues may comprise a cysteine. Thetwo or more residues in the peptide region may comprise cysteine. Insome instances, at least one of the two or more residues is notcysteine. In some instances, at least two of the two or more residuesare not cysteine. The one or more TAs may comprise GLP-1, GLP-2,Exendin-4, exenatide, oxyntomodulin, glucagon, FGF21, a GLP-1R and GIPRdual agonist, a GLP-1R and GCGR dual agonist, or derivative thereof. TheGLP-1 may be a human GLP-1. The FGF21 may be a human FGF21. The one ormore lipids may comprise one or more sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,and fatty alcohols. The metabolic disease or condition may be diabetes.The metabolic disease or condition may be obesity. The metabolic diseaseor condition may be glycogen storage disease, phenylketonuria, maplesyrup urine disease, glutaric acidemia type 1, Carbamoyl phosphatesynthetase I deficiency, alcaptonuria, Medium-chain acyl-coenzyme Adehydrogenase deficiency (MCADD), acute intermittent porphyria,Lesch-Nyhan syndrome, lipoid congenital adrenal hyperplasia, congenitaladrenal hyperplasia, Kearns-Sayre syndrome, Zellweger syndrome,Gaucher's disease, or Niemann Pick disease.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more PLCs, wherein the one or morepeptide lipid conjugates (PLCs) comprise (a) one or more lipids; and (b)one or more peptide conjugates (PC), the peptide conjugate (PC)comprising a peptide region comprising one or more peptide therapeuticagents (TA) and a staple region comprising one or more staples, the oneor more staples connect two or more residues in the peptide region,wherein the one or more lipids are attached to the one or more staples.At least one of the two or more residues may comprise a cysteine. Thetwo or more residues in the peptide region may comprise cysteine. Insome instances, at least one of the two or more residues is notcysteine. In some instances, at least two of the two or more residuesare not cysteine. The one or more TAs may comprise GLP-1, GLP-2,Exendin-4, exenatide, oxyntomodulin, glucagon, FGF21, a GLP-1R and GIPRdual agonist, a GLP-1R and GCGR dual agonist, or derivative thereof. TheGLP-1 may be a human GLP-1. The FGF21 may be a human FGF21. The one ormore lipids may comprise one or more sterols, sterol derivatives, bileacids, vitamin E derivatives, fatty di-acids, fatty acids, fatty amides,and fatty alcohols. The metabolic disease or condition may be diabetes.The metabolic disease or condition may be obesity. The metabolic diseaseor condition may be glycogen storage disease, phenylketonuria, maplesyrup urine disease, glutaric acidemia type 1, Carbamoyl phosphatesynthetase I deficiency, alcaptonuria, Medium-chain acyl-coenzyme Adehydrogenase deficiency (MCADD), acute intermittent porphyria,Lesch-Nyhan syndrome, lipoid congenital adrenal hyperplasia, congenitaladrenal hyperplasia, Kearns-Sayre syndrome, Zellweger syndrome,Gaucher's disease, or Niemann Pick disease.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more PLCs, wherein the one or morepeptide lipid conjugates (PLCs) comprise (a) two or more lipids; and (b)one or more peptide conjugates (PC), the peptide conjugate (PC)comprising a peptide region comprising one or more peptide therapeuticagents (TA) and a staple region comprising one or more staples, the oneor more staples connect two or more residues in the peptide region,wherein at least one of the two or more lipids are attached to the oneor more therapeutic agents and at least one of the two or more lipidsare attached to the one or more staples. At least one of the two or moreresidues may comprise a cysteine. The two or more residues in thepeptide region may comprise cysteine. In some instances, at least one ofthe two or more residues is not cysteine. In some instances, at leasttwo of the two or more residues are not cysteine. The one or more TAsmay comprise GLP-1, GLP-2, Exendin-4, exenatide, oxyntomodulin,glucagon, FGF21, a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dualagonist, or derivative thereof. The GLP-1 may be a human GLP-1. TheFGF21 may be a human FGF21. The one or more lipids may comprise one ormore sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, and fatty alcohols. Themetabolic disease or condition may be diabetes. The metabolic disease orcondition may be obesity. The metabolic disease or condition may beglycogen storage disease, phenylketonuria, maple syrup urine disease,glutaric acidemia type 1, Carbamoyl phosphate synthetase I deficiency,alcaptonuria, Medium-chain acyl-coenzyme A dehydrogenase deficiency(MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipoidcongenital adrenal hyperplasia, congenital adrenal hyperplasia,Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or NiemannPick disease.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more peptide lipid conjugates (PLCs)of Formula (II), Formula (III), or Formula (IV). The one or more lipidsmay be attached to the one or more TAs. The one or more lipids may beattached to the one or more staples. At least one of the two or moreresidues may comprise a cysteine. The two or more residues in thepeptide region may comprise cysteine. In some instances, at least one ofthe two or more residues is not cysteine. In some instances, at leasttwo of the two or more residues are not cysteine. The one or more TAsmay comprise GLP-1, GLP-2, Exendin-4, exenatide, oxyntomodulin,glucagon, FGF21, a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dualagonist, or derivative thereof. The GLP-1 may be a human GLP-1. TheFGF21 may be a human FGF21. The one or more lipids may comprise one ormore sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, and fatty alcohols. Themetabolic disease or condition may be diabetes. The metabolic disease orcondition may be obesity. The metabolic disease or condition may beglycogen storage disease, phenylketonuria, maple syrup urine disease,glutaric acidemia type 1, Carbamoyl phosphate synthetase I deficiency,alcaptonuria, Medium-chain acyl-coenzyme A dehydrogenase deficiency(MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipoidcongenital adrenal hyperplasia, congenital adrenal hyperplasia,Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or NiemannPick disease.

Provided herein is a method of preventing or treating NAFLD, NASH, orcardiovascular disease in a subject in need thereof comprisingadministering to the subject one or more PLCs, wherein the one or morepeptide lipid conjugates (PLCs) comprise (a) one or more lipids; and (b)one or more peptide conjugates (PC), the peptide conjugate (PC)comprising a peptide region comprising one or more peptide therapeuticagents (TA) and a staple region comprising one or more staples, the oneor more staples connect two or more residues in the peptide region,wherein the one or more lipids are attached to the one or more peptideconjugates The one or more lipids may be attached to the one or moreTAs. The one or more lipids may be attached to the one or more staples.At least one of the two or more residues may comprise a cysteine. Thetwo or more residues in the peptide region may comprise cysteine. Insome instances, at least one of the two or more residues is notcysteine. In some instances, at least two of the two or more residuesare not cysteine. The one or more lipids may comprise one or moresterols, sterol derivatives, bile acids, vitamin E derivatives, fattydi-acids, fatty acids, fatty amides, and fatty alcohols. The one or moreTAs may comprise GLP-1, GLP-2, Exendin-4, exenatide, oxyntomodulin,glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R and GCGR dualagonist, or derivative thereof. The GLP-1 may be a human GLP-1.

Provided herein is a method of preventing or treating NAFLD, NASH, orcardiovascular disease in a subject in need thereof comprisingadministering to the subject one or more PLCs, wherein the one or morepeptide lipid conjugates (PLCs) comprise (a) one or more lipids; and (b)one or more peptide conjugates (PC), the peptide conjugate (PC)comprising a peptide region comprising one or more peptide therapeuticagents (TA) and a staple region comprising one or more staples, the oneor more staples connect two or more residues in the peptide region,wherein the one or more lipids are attached to the one or more staples.The one or more lipids may be attached to the one or more TAs. At leastone of the two or more residues may comprise a cysteine. The two or moreresidues in the peptide region may comprise cysteine. In some instances,at least one of the two or more residues is not cysteine. In someinstances, at least two of the two or more residues are not cysteine.The one or more lipids may comprise one or more sterols, sterolderivatives, bile acids, vitamin E derivatives, fatty di-acids, fattyacids, fatty amides, and fatty alcohols. The one or more TAs maycomprise GLP-1, GLP-2, Exendin-4, exenatide, oxyntomodulin, glucagon, aGLP-1R and GIPR dual agonist, a GLP-1R and GCGR dual agonist, orderivative thereof. The GLP-1 may be a human GLP-1.

Provided herein is a method of preventing or treating NAFLD, NASH, orcardiovascular disease in a subject in need thereof comprisingadministering to the subject one or more PLCs, wherein the one or morepeptide lipid conjugates (PLCs) comprise (a) two or more lipids; and (b)one or more peptide conjugates (PC), the peptide conjugate (PC)comprising a peptide region comprising one or more peptide therapeuticagents (TA) and a staple region comprising one or more staples, the oneor more staples connect two or more residues in the peptide region,wherein at least one of the two or more lipids are attached to the oneor more therapeutic agents and at least one of the two or more lipidsare attached to the one or more staples. The one or more lipids may beattached to the one or more TAs. At least one of the two or moreresidues may comprise a cysteine. The two or more residues in thepeptide region may comprise cysteine. In some instances, at least one ofthe two or more residues is not cysteine. In some instances, at leasttwo of the two or more residues are not cysteine. The one or more lipidsmay comprise one or more sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols. The one or more TAs may comprise GLP-1, GLP-2,Exendin-4, exenatide, oxyntomodulin, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, or derivative thereof. TheGLP-1 may be a human GLP-1.

Provided herein is a method of preventing or treating NAFLD, NASH, orcardiovascular disease in a subject in need thereof comprisingadministering to the subject one or more peptide lipid conjugates (PLCs)of Formula (II), Formula (III), or Formula (IV). At least one of the twoor more residues may comprise a cysteine. The two or more residues inthe peptide region may comprise cysteine. In some instances, at leastone of the two or more residues is not cysteine. In some instances, atleast two of the two or more residues are not cysteine. The one or morelipids may comprise one or more sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols. The one or more lipids may comprise one or more sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols. The one or more TAs maycomprise GLP-1, GLP-2, Exendin-4, exenatide, oxyntomodulin, glucagon, aGLP-1R and GIPR dual agonist, a GLP-1R and GCGR dual agonist, orderivative thereof. The GLP-1 may be a human GLP-1.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids; and (b) one or morepeptide conjugates (PC), the peptide conjugate (PC) comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more peptide conjugates. The oneor more lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. At least one of thetwo or more residues may comprise a cysteine. The two or more residuesin the peptide region may comprise cysteine. In some instances, at leastone of the two or more residues is not cysteine. In some instances, atleast two of the two or more residues are not cysteine. The one or morelipids may comprise one or more sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols. The one or more TAs may comprise GLP-1, GLP-2,Exendin-4, exenatide, oxyntomodulin, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, or derivative thereof. TheGLP-1 may be a human GLP-1. The disease or condition may be a metabolicdisease or disorder. The disease or condition may be diabetes. Thedisease or condition may be obesity. Additional diseases and/orconditions which benefit from a GLP-1R and/or GCGR agonist include, butare not limited to, dyslipidemia, cardiovascular and fatty liverdiseases.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) one or more lipids; and (b) one or morepeptide conjugates (PC), the peptide conjugate (PC) comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more staples. The one or morelipids may be attached to the one or more TAs. At least one of the twoor more residues may comprise a cysteine. The two or more residues inthe peptide region may comprise cysteine. In some instances, at leastone of the two or more residues is not cysteine. In some instances, atleast two of the two or more residues are not cysteine. The one or morelipids may comprise one or more sterols, sterol derivatives, bile acids,vitamin E derivatives, fatty di-acids, fatty acids, fatty amides, andfatty alcohols. The one or more TAs may comprise GLP-1, GLP-2,Exendin-4, exenatide, oxyntomodulin, glucagon, a GLP-1R and GIPR dualagonist, a GLP-1R and GCGR dual agonist, or derivative thereof. TheGLP-1 may be a human GLP-1. The disease or condition may be a metabolicdisease or disorder. The disease or condition may be diabetes. Thedisease or condition may be obesity. Additional diseases and/orconditions which benefit from a GLP-1R and/or GCGR agonist include, butare not limited to, dyslipidemia, cardiovascular and fatty liverdiseases.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more PLCs, wherein the one or more peptide lipidconjugates (PLCs) comprise (a) two or more lipids; and (b) one or morepeptide conjugates (PC), the peptide conjugate (PC) comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein at least oneof the two or more lipids are attached to the one or more therapeuticagents and at least one of the two or more lipids are attached to theone or more staples. At least one of the two or more residues maycomprise a cysteine. The two or more residues in the peptide region maycomprise cysteine. In some instances, at least one of the two or moreresidues is not cysteine. In some instances, at least two of the two ormore residues are not cysteine. The one or more lipids may comprise oneor more sterols, sterol derivatives, bile acids, vitamin E derivatives,fatty di-acids, fatty acids, fatty amides, and fatty alcohols. The oneor more TAs may comprise GLP-1, GLP-2, Exendin-4, exenatide,oxyntomodulin, glucagon, a GLP-1R and GIPR dual agonist, a GLP-1R andGCGR dual agonist, or derivative thereof. The GLP-1 may be a humanGLP-1. The disease or condition may be a metabolic disease or disorder.The disease or condition may be diabetes. The disease or condition maybe obesity. Additional diseases and/or conditions which benefit from aGLP-1R and/or GCGR agonist include, but are not limited to,dyslipidemia, cardiovascular and fatty liver diseases.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more peptide lipid conjugates (PLCs) of Formula (II),Formula (III), or Formula (IV). At least one of the two or more residuesmay comprise a cysteine. The two or more residues in the peptide regionmay comprise cysteine. In some instances, at least one of the two ormore residues is not cysteine. In some instances, at least two of thetwo or more residues are not cysteine. The one or more lipids maycomprise one or more sterols, sterol derivatives, bile acids, vitamin Ederivatives, fatty di-acids, fatty acids, fatty amides, and fattyalcohols. The one or more lipids may comprise one or more sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols. The one or more TAs maycomprise GLP-1, GLP-2, Exendin-4, exenatide, oxyntomodulin, glucagon, aGLP-1R and GIPR dual agonist, a GLP-1R and GCGR dual agonist, orderivative thereof. The GLP-1 may be a human GLP-1. The disease orcondition may be a metabolic disease or disorder. The disease orcondition may be diabetes. The disease or condition may be obesity.Additional diseases and/or conditions which benefit from a GLP-1R and/orGCGR agonist include, but are not limited to, dyslipidemia,cardiovascular and fatty liver diseases.

Disclosed herein are methods of treating a disease or condition in asubject in need thereof, the method comprising administering to thesubject a composition disclosed herein comprising one or more mTAs.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject a composition comprising a mTA, wherein the mTA comprises atherapeutic agent (TA), a staple, and a half-life extending molecule(HEM). The disease or condition may be a diabetes or obesity, or amedical condition associated with diabetes or obesity. The disease orcondition may be non-alcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), or cardiovascular disease. The disease orcondition may be short bowel syndrome (SBS). The disease or conditionmay be inflammatory bowel disease (IBD), inflammatory bowel syndrome(IBS), or psoriasis. The disease or condition may be Crohn's disease orulcerative colitis. The disease or condition may be Alzheimer's disease,Parkinson's disease or Huntington's disease. The PLC may be administeredwith one or more additional therapeutic agents. The additionaltherapeutic agents may comprise one or more other diabetes drugs, DPP4inhibitors, SGLT2 inhibitors, hypoglycemic drugs and biguanidine drugs,insulin secretogogues and sulfonyl urea drugs, TZD drugs, insulin andinsulin analogs, FGF21 and analogs, leptin or leptin analogs, amylin andamylin analogs, an anti-inflammatory drug, cyclosporine A or FK506,5-ASA, or a statin, or any combination thereof. The additionaltherapeutic agent may be aspirin.

Further disclosed herein are methods for treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more mTAs, wherein each of the one or more mTAs comprisea therapeutic agent (TA), a staple, and a half-life extending molecule(HEM). The disease or condition may be a diabetes or obesity, or amedical condition associated with diabetes or obesity. The disease orcondition may be non-alcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), or cardiovascular disease. The disease orcondition may be short bowel syndrome (SBS). The disease or conditionmay be inflammatory bowel disease (IBD), inflammatory bowel syndrome(IBS), or psoriasis. The disease or condition may be Crohn's disease orulcerative colitis. The disease or condition may be Alzheimer's disease,Parkinson's disease or Huntington's disease. The PLC may be administeredwith one or more additional therapeutic agents. The additionaltherapeutic agents may comprise one or more other diabetes drugs, DPP4inhibitors, SGLT2 inhibitors, hypoglycemic drugs and biguanidine drugs,insulin secretogogues and sulfonyl urea drugs, TZD drugs, insulin andinsulin analogs, FGF21 and analogs, leptin or leptin analogs, amylin andamylin analogs, an anti-inflammatory drug, cyclosporine A or FK506,5-ASA, or a statin, or any combination thereof. The additionaltherapeutic agent may be aspirin.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more mTAs, wherein each of the one or more mTAs comprisesa therapeutic agent (TA), a first staple, and a first half-lifeextending molecule (HEM); wherein the therapeutic agent is a modified orunmodified therapeutic peptide that is covalently attached to the firststaple via two amino acid residues on the modified or unmodifiedtherapeutic peptide. The disease or condition may be a diabetes orobesity, or a medical condition associated with diabetes or obesity. Thedisease or condition may be non-alcoholic fatty liver disease (NAFLD),nonalcoholic steatohepatitis (NASH), or cardiovascular disease. Thedisease or condition may be short bowel syndrome (SBS). The disease orcondition may be inflammatory bowel disease (IBD), inflammatory bowelsyndrome (IBS), or psoriasis. The disease or condition may be Crohn'sdisease or ulcerative colitis. The disease or condition may beAlzheimer's disease, Parkinson's disease or Huntington's disease. ThePLC may be administered with one or more additional therapeutic agents.The additional therapeutic agents may comprise one or more otherdiabetes drugs, DPP4 inhibitors, SGLT2 inhibitors, hypoglycemic drugsand biguanidine drugs, insulin secretogogues and sulfonyl urea drugs,TZD drugs, insulin and insulin analogs, FGF21 and analogs, leptin orleptin analogs, amylin and amylin analogs, an anti-inflammatory drug,cyclosporine A or FK506, 5-ASA, or a statin, or any combination thereof.The additional therapeutic agent may be aspirin.

Further disclosed herein are methods of treating a disease or conditionin a subject in need thereof, the method comprising administering to thesubject one or more mTAs, wherein each of the one or more mTAs comprisesa therapeutic agent (TA), a first staple, and a first half-lifeextending molecule (HEM); wherein the therapeutic agent is a modified orunmodified therapeutic peptide that is covalently attached to the firststaple via two amino acid residues on the modified or unmodifiedtherapeutic peptide and the first HEM is covalently attached to thefirst staple. The disease or condition may be a diabetes or obesity, ora medical condition associated with diabetes or obesity. The disease orcondition may be non-alcoholic fatty liver disease (NAFLD), nonalcoholicsteatohepatitis (NASH), or cardiovascular disease. The disease orcondition may be short bowel syndrome (SBS). The disease or conditionmay be inflammatory bowel disease (IBD), inflammatory bowel syndrome(IBS), or psoriasis. The disease or condition may be Crohn's disease orulcerative colitis. The disease or condition may be Alzheimer's disease,Parkinson's disease or Huntington's disease. The PLC may be administeredwith one or more additional therapeutic agents. The additionaltherapeutic agents may comprise one or more other diabetes drugs, DPP4inhibitors, SGLT2 inhibitors, hypoglycemic drugs and biguanidine drugs,insulin secretogogues and sulfonyl urea drugs, TZD drugs, insulin andinsulin analogs, FGF21 and analogs, leptin or leptin analogs, amylin andamylin analogs, an anti-inflammatory drug, cyclosporine A or FK506,5-ASA, or a statin, or any combination thereof. The additionaltherapeutic agent may be aspirin.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof, the method comprisingadministering to the subject a composition disclosed herein comprisingone or more mTAs.

Provided herein is a method of preventing or treating a metabolicdisease or condition in a subject in need thereof comprisingadministering to the subject one or more mTAs, wherein each of the oneor more mTAs comprises a therapeutic agent (TA), a first staple, and afirst half-life extending molecule (HEM); wherein the therapeutic agentis a modified or unmodified therapeutic peptide that is covalentlyattached to the first staple via two amino acid residues on the modifiedor unmodified therapeutic peptide and the first HEM is covalentlyattached to the first staple. The metabolic disease or condition may bediabetes. The metabolic disease or condition may be obesity. Themetabolic disease or condition may be glycogen storage disease,phenylketonuria, maple syrup urine disease, glutaric acidemia type 1,Carbamoyl phosphate synthetase I deficiency, alcaptonuria, Medium-chainacyl-coenzyme A dehydrogenase deficiency (MCADD), acute intermittentporphyria, Lesch-Nyhan syndrome, lipoid congenital adrenal hyperplasia,congenital adrenal hyperplasia, Kearns-Sayre syndrome, Zellwegersyndrome, Gaucher's disease, or Niemann Pick disease.

Provided herein is a method of preventing or treating NAFLD, NASH, orcardiovascular disease in a subject in need thereof, the methodcomprising administering to the subject a composition disclosed hereincomprising one or more mTAs.

Provided herein is a method of preventing or treating NAFLD, NASH, orcardiovascular disease in a subject in need thereof comprisingadministering to the subject one or more mTAs, wherein each of the oneor more mTAs comprises a therapeutic agent (TA), a first staple, and afirst half-life extending molecule (HEM); wherein the therapeutic agentis a modified or unmodified therapeutic peptide that is covalentlyattached to the first staple via two amino acid residues on the modifiedor unmodified therapeutic peptide and the first HEM is covalentlyattached to the first staple.

Provided herein is a method of preventing or treating short bowelsyndrome (SBS) in a subject in need thereof, the method comprisingadministering to the subject a composition disclosed herein comprisingone or more mTAs.

Provided herein is a method of preventing or treating short bowelsyndrome (SBS) in a subject in need thereof comprising administering tothe subject one or more mTAs, wherein each of the one or more mTAscomprises a therapeutic agent (TA), a first staple, and a firsthalf-life extending molecule (HEM); wherein the therapeutic agent is amodified or unmodified therapeutic peptide that is covalently attachedto the first staple via two amino acid residues on the modified orunmodified therapeutic peptide and the first HEM is covalently attachedto the first staple.

Provided herein is a method of preventing or treating inflammatory boweldisease (IBD), inflammatory bowel syndrome (IBS), or psoriasis in asubject in need thereof, the method comprising administering to thesubject a composition disclosed herein comprising one or more mTAs.

Provided herein is a method of preventing or treating inflammatory boweldisease (IBD), inflammatory bowel syndrome (IBS), or psoriasis in asubject in need thereof comprising administering to the subject one ormore mTAs, wherein each of the one or more mTAs comprises a therapeuticagent (TA), a first staple, and a first half-life extending molecule(HEM); wherein the therapeutic agent is a modified or unmodifiedtherapeutic peptide that is covalently attached to the first staple viatwo amino acid residues on the modified or unmodified therapeuticpeptide and the first HEM is covalently attached to the first staple.

Provided herein is a method of preventing or treating Crohn's disease orulcerative colitis in a subject in need thereof, the method comprisingadministering to the subject a composition disclosed herein comprisingone or more mTAs.

Provided herein is a method of preventing or treating Crohn's disease orulcerative colitis in a subject in need thereof comprising administeringto the subject one or more mTAs, wherein each of the one or more mTAscomprises a therapeutic agent (TA), a first staple, and a firsthalf-life extending molecule (HEM); wherein the therapeutic agent is amodified or unmodified therapeutic peptide that is covalently attachedto the first staple via two amino acid residues on the modified orunmodified therapeutic peptide and the first HEM is covalently attachedto the first staple.

Provided herein is a method of preventing or treating Alzheimer'sdisease, Parkinson's disease or Huntington's disease in a subject inneed thereof, the method comprising administering to the subject acomposition disclosed herein comprising one or more mTAs.

Provided herein is a method of preventing or treating Alzheimer'sdisease, Parkinson's disease or Huntington's disease in a subject inneed thereof comprising administering to the subject one or more mTAs,wherein each of the one or more mTAs comprises a therapeutic agent (TA),a first staple, and a first half-life extending molecule (HEM); whereinthe therapeutic agent is a modified or unmodified therapeutic peptidethat is covalently attached to the first staple via two amino acidresidues on the modified or unmodified therapeutic peptide and the firstHEM is covalently attached to the first staple.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more mTAs disclosed herein.

Provided herein is a method of preventing or treating a disease orcondition which benefits from a GLP-1R and/or glucagon receptor (GCGR)agonist in a subject in need thereof comprising administering to thesubject one or more mTAs, wherein each of the one or more mTAs comprisesa therapeutic agent (TA), a first staple, and a first half-lifeextending molecule (HEM); wherein the therapeutic agent is a modified orunmodified therapeutic peptide that is covalently attached to the firststaple via two amino acid residues on the modified or unmodifiedtherapeutic peptide and the first HEM is covalently attached to thefirst staple. The disease or condition may be a metabolic disease ordisorder. The disease or condition may be diabetes. The disease orcondition may be obesity. Additional diseases and/or conditions whichbenefit from a GLP-1R and/or GCGR agonist include, but are not limitedto, dyslipidemia, cardiovascular and fatty liver diseases.

Compositions

Disclosed herein are pharmaceutical compositions comprising a PLCdisclosed herein. The compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more PLCs. The PLCs may be different. Alternatively, the PLCsmay be the same or similar. The compositions may comprise 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more PLCs. The PLCs may be different. The PLCs maycomprise different therapeutic agents, different lipids, or acombination thereof. The PLCs may be the same or similar.

Disclosed herein are pharmaceutical compositions comprising a mTAdisclosed herein. The compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8,9, 10 or more mTAs. The mTAs may be different. Alternatively, the mTAsmay be the same or similar. The compositions may comprise 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more mTAs. The mTAs may be different. The mTAs maycomprise different therapeutic agents, different HEMs, or a combinationthereof. The mTAs may be the same or similar.

Further disclosed herein are compositions comprising one or more mTAs,wherein each of the one or more mTAs comprises a therapeutic agent (TA),a staple, and a half-life extending molecule (HEM). The TA may be amodified or unmodified therapeutic peptide. The TA may be covalentlyattached to the staple via two amino acid residues on the modified orunmodified therapeutic peptide. At least one of the two amino acidresidues may comprise a cysteine. The composition may further compriseone or more pharmaceutically acceptable salts, excipients or vehicles.

Further disclosed herein are compositions comprising one or more mTAs,wherein each of the one or more mTAs comprises a therapeutic agent (TA),a first staple, and a first half-life extending molecule (HEM); whereinthe therapeutic agent is a modified or unmodified therapeutic peptidethat is covalently attached to the first staple via two amino acidresidues on the modified or unmodified therapeutic peptide. At least oneof the two amino acid residues may comprise a cysteine. The first HEMmay be covalently attached to the first staple. The first HEM may becovalently attached to the TA. The composition may further comprise oneor more pharmaceutically acceptable salts, excipients or vehicles.

Further disclosed herein are compositions comprising one or more mTAs,wherein each of the one or more mTAs comprises a therapeutic agent (TA),a first staple, and a first half-life extending molecule (HEM); whereinthe therapeutic agent is a modified or unmodified therapeutic peptidethat is covalently attached to the first staple via two amino acidresidues on the modified or unmodified therapeutic peptide and the firstHEM is covalently attached to the first staple. At least one of the twoamino acid residues may comprise a cysteine. The composition may furthercomprise one or more pharmaceutically acceptable salts, excipients orvehicles.

Further disclosed herein are compositions comprising one or more mTAs,wherein each of the one or more mTAs comprises a therapeutic agent (TA),a first staple, and a first half-life extending molecule (HEM); whereinthe therapeutic agent is a modified or unmodified therapeutic peptidethat is covalently attached to the first staple via two amino acidresidues on the modified or unmodified therapeutic peptide; theunmodified therapeutic peptide is selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP; the modified therapeuticpeptide is a derivative of a peptide selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof, and the first HEM is covalentlyattached to the first staple. At least one of the two amino acidresidues may comprise a cysteine. The composition may further compriseone or more pharmaceutically acceptable salts, excipients or vehicles.

Further disclosed herein are compositions comprising one or more mTAs,wherein each of the one or more mTAs comprises a therapeutic agent (TA),a first staple, and a first half-life extending molecule (HEM); whereinthe therapeutic agent is a modified or unmodified therapeutic peptidethat is covalently attached to the first staple via two amino acidresidues on the modified or unmodified therapeutic peptide; the firstHEM is covalently attached to the first staple; and the first HEMcomprises a lipid, a polyglycol region, or a combination thereof. Atleast one of the two amino acid residues may comprise a cysteine. Thecomposition may further comprise one or more pharmaceutically acceptablesalts, excipients or vehicles.

Further disclosed herein are compositions comprising one or more mTAs,wherein each of the one or more mTAs comprises a therapeutic agent (TA),a first staple, and a first half-life extending molecule (HEM); whereinthe therapeutic agent is a modified or unmodified therapeutic peptidethat is covalently attached to the first staple via two amino acidresidues on the modified or unmodified therapeutic peptide; theunmodified therapeutic peptide is selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP; the modified therapeuticpeptide is a derivative of a peptide selected from GLP-1, glucagon,oxyntomodulin, exendin-4, GLP-2, and GIP, the derivative being a peptidecomprising one or more amino acid additions, deletions, orsubstitutions, or a combination thereof; the first HEM is covalentlyattached to the first staple; and the first HEM comprises a lipid, apolyglycol region, or a combination thereof. At least one of the twoamino acid residues may comprise a cysteine. The composition may furthercomprise one or more pharmaceutically acceptable salts, excipients orvehicles.

Disclosed herein are compositions comprising one or more PLCs, whereinthe one or more peptide lipid conjugates (PLCs) comprise (a) one or morelipids; and (b) one or more peptide conjugates (PC), the peptideconjugate (PC) comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, wherein the one or more lipids are attached to theone or more peptide conjugates. The one or more lipids may be attachedto the one or more TAs. The one or more lipids may be attached to theone or more staples. At least one of the two or more residues maycomprise a cysteine. The two or more residues in the peptide region maycomprise cysteine. In some instances, at least one of the two or moreresidues is not cysteine. In some instances, at least two of the two ormore residues are not cysteine. The composition may further comprise oneor more pharmaceutically acceptable salts, excipients or vehicles.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) oneor more lipids, the lipids selected from a group consisting of sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or morepeptide conjugates (PC), the peptide conjugate comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more peptide conjugates. The oneor more lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. At least one of thetwo or more residues may comprise a cysteine. The two or more residuesin the peptide region may comprise cysteine. In some instances, at leastone of the two or more residues is not cysteine. In some instances, atleast two of the two or more residues are not cysteine. The compositionmay further comprise one or more pharmaceutically acceptable salts,excipients or vehicles.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) oneor more lipids; and (b) one or more peptide conjugates (PC), wherein thepeptide conjugate comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, the one or more peptide therapeutic agentscomprising one or more oxyntomodulin, exenatide, exendin-4,glucagon-like protein-1 (GLP-1), GLP-2, a GLP-1R and GIPR dual agonist,a GLP-1R and GCGR dual agonist, or glucagon, wherein the one or morelipids are attached to the one or more peptide conjugates. The one ormore lipids may be attached to the one or more TAs. The one or morelipids may be attached to the one or more staples. At least one of thetwo or more residues may comprise a cysteine. The two or more residuesin the peptide region may comprise cysteine. In some instances, at leastone of the two or more residues is not cysteine. In some instances, atleast two of the two or more residues are not cysteine. The compositionmay further comprise one or more pharmaceutically acceptable salts,excipients or vehicles.

Disclosed herein are compositions comprising one or more PLCs, whereinthe one or more peptide lipid conjugates (PLCs) comprise (a) one or morelipids; and (b) one or more peptide conjugates (PC), the peptideconjugate (PC) comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, wherein the one or more lipids are attached to theone or more staples. The one or more lipids may be attached to the oneor more TAs. At least one of the two or more residues may comprise acysteine. The two or more residues in the peptide region may comprisecysteine. In some instances, at least one of the two or more residues isnot cysteine. In some instances, at least two of the two or moreresidues are not cysteine. The composition may further comprise one ormore pharmaceutically acceptable salts, excipients or vehicles.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) oneor more lipids, the lipids selected from a group consisting of sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or morepeptide conjugates (PC), the peptide conjugate comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein the one ormore lipids are attached to the one or more staples. The one or morelipids may be attached to the one or more TAs. At least one of the twoor more residues may comprise a cysteine. The two or more residues inthe peptide region may comprise cysteine. In some instances, at leastone of the two or more residues is not cysteine. In some instances, atleast two of the two or more residues are not cysteine. The compositionmay further comprise one or more pharmaceutically acceptable salts,excipients or vehicles.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) oneor more lipids; and (b) one or more peptide conjugates (PC), wherein thepeptide conjugate comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, the one or more peptide therapeutic agentscomprising one or more oxyntomodulin, exenatide, exendin-4,glucagon-like protein-1 (GLP-1), GLP-2, a GLP-1R and GIPR dual agonist,a GLP-1R and GCGR dual agonist, or glucagon, wherein the one or morelipids are attached to the one or more staples. The one or more lipidsmay be attached to the one or more TAs. The one or more lipids may beattached to the one or more staples. At least one of the two or moreresidues may comprise a cysteine. The two or more residues in thepeptide region may comprise cysteine. In some instances, at least one ofthe two or more residues is not cysteine. In some instances, at leasttwo of the two or more residues are not cysteine. The composition mayfurther comprise one or more pharmaceutically acceptable salts,excipients or vehicles.

Disclosed herein are compositions comprising one or more PLCs, whereinthe one or more peptide lipid conjugates (PLCs) comprise (a) two or morelipids; and (b) one or more peptide conjugates (PC), the peptideconjugate (PC) comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, wherein at least one of the two or more lipids areattached to the one or more therapeutic agents and at least one of thetwo or more lipids are attached to the one or more staples. The one ormore lipids may be attached to the one or more TAs. At least one of thetwo or more residues may comprise a cysteine. The two or more residuesin the peptide region may comprise cysteine. In some instances, at leastone of the two or more residues is not cysteine. In some instances, atleast two of the two or more residues are not cysteine. The compositionmay further comprise one or more pharmaceutically acceptable salts,excipients or vehicles.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) twoor more lipids, the lipids selected from a group consisting of sterols,sterol derivatives, bile acids, vitamin E derivatives, fatty di-acids,fatty acids, fatty amides, and fatty alcohols; and (b) one or morepeptide conjugates (PC), the peptide conjugate comprising a peptideregion comprising one or more peptide therapeutic agents (TA) and astaple region comprising one or more staples, the one or more staplesconnect two or more residues in the peptide region, wherein at least oneof the two or more lipids are attached to the one or more therapeuticagents and at least one of the two or more lipids are attached to theone or more staples. The one or more lipids may be attached to the oneor more TAs. At least one of the two or more residues may comprise acysteine. The two or more residues in the peptide region may comprisecysteine. In some instances, at least one of the two or more residues isnot cysteine. In some instances, at least two of the two or moreresidues are not cysteine. The composition may further comprise one ormore pharmaceutically acceptable salts, excipients or vehicles.

Further disclosed herein are compositions comprising one or more PLCs,wherein the one or more peptide lipid conjugates (PLCs) comprise (a) twoor more lipids; and (b) one or more peptide conjugates (PC), wherein thepeptide conjugate comprising a peptide region comprising one or morepeptide therapeutic agents (TA) and a staple region comprising one ormore staples, the one or more staples connect two or more residues inthe peptide region, the one or more peptide therapeutic agentscomprising one or more oxyntomodulin, exenatide, exendin-4,glucagon-like protein-1 (GLP-1), GLP-2, a GLP-1R and GIPR dual agonist,a GLP-1R and GCGR dual agonist, or glucagon, wherein at least one of thetwo or more lipids are attached to the one or more therapeutic agentsand at least one of the two or more lipids are attached to the one ormore staples. The one or more lipids may be attached to the one or moreTAs. At least one of the two or more residues may comprise a cysteine.The two or more residues in the peptide region may comprise cysteine. Insome instances, at least one of the two or more residues is notcysteine. In some instances, at least two of the two or more residuesare not cysteine. The composition may further comprise one or morepharmaceutically acceptable salts, excipients or vehicles.

Further disclosed herein are compositions comprising one or more peptidelipid conjugates (PLCs) of Formula (II), Formula (III), or Formula (IV).The one or more lipids may be attached to the one or more TAs. The oneor more lipids may be attached to the one or more staples. At least oneof the two or more residues may comprise a cysteine. The two or moreresidues in the peptide region may comprise cysteine. In some instances,at least one of the two or more residues is not cysteine. In someinstances, at least two of the two or more residues are not cysteine.

The compositions disclosed herein may further comprise one or morepharmaceutically acceptable salts, excipients or vehicles.Pharmaceutically acceptable salts, excipients, or vehicles may includecarriers, excipients, diluents, antioxidants, preservatives, coloring,flavoring and diluting agents, emulsifying agents, suspending agents,solvents, fillers, bulking agents, buffers, delivery vehicles, tonicityagents, cosolvents, wetting agents, complexing agents, buffering agents,antimicrobials, and surfactants.

Neutral buffered saline or saline mixed with serum albumin are exemplaryappropriate carriers. The pharmaceutical compositions may includeantioxidants such as ascorbic acid; low molecular weight polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, arginine or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugar alcohols such as mannitolor sorbitol; salt-forming counterions such as sodium; and/or nonionicsurfactants such as Tween, pluronics, or polyethylene glycol (PEG). Alsoby way of example, suitable tonicity enhancing agents include alkalimetal halides (preferably sodium or potassium chloride), mannitol,sorbitol, and the like. Suitable preservatives include benzalkoniumchloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid and the like. Hydrogen peroxide also may beused as preservative. Suitable cosolvents include glycerin, propyleneglycol, and PEG. Suitable complexing agents include caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agentsinclude sorbitan esters, polysorbates such as polysorbate 80,tromethamine, lecithin, cholesterol, tyloxapal, and the like. Thebuffers may be conventional buffers such as acetate, borate, citrate,phosphate, bicarbonate, or Tris-HCl. Acetate buffer may be about pH4-5.5, and Tris buffer can be about pH 7-8.5. Additional pharmaceuticalagents are set forth in Remington's Pharmaceutical Sciences, 18thEdition, A. R. Gennaro, ed., Mack Publishing Company, 1990.

The composition may be in liquid form or in a lyophilized orfreeze-dried form and may include one or more lyoprotectants,excipients, surfactants, high molecular weight structural additivesand/or bulking agents (see, for example, U.S. Pat. Nos. 6,685,940,6,566,329, and 6,372,716). In one embodiment, a lyoprotectant isincluded, which is a non-reducing sugar such as sucrose, lactose ortrehalose. The amount of lyoprotectant generally included is such that,upon reconstitution, the resulting formulation will be isotonic,although hypertonic or slightly hypotonic formulations also may besuitable. In addition, the amount of lyoprotectant should be sufficientto prevent an unacceptable amount of degradation and/or aggregation ofthe protein upon lyophilization. Exemplary lyoprotectant concentrationsfor sugars (e.g., sucrose, lactose, trehalose) in the pre-lyophilizedformulation are from about 10 mM to about 400 mM. In another embodiment,a surfactant is included, such as for example, nonionic surfactants andionic surfactants such as polysorbates (e.g., polysorbate 20,polysorbate 80); poloxamers (e.g., poloxamer 188); poly(ethylene glycol)phenyl ethers (e.g., Triton); sodium dodecyl sulfate (SDS); sodiumlaurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-,or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl ofeyl-taurate; and the MONAQUAT™. series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., Pluronics, PF68 etc). Exemplaryamounts of surfactant that may be present in the pre-lyophilizedformulation are from about 0.001-0.5%. High molecular weight structuraladditives (e.g., fillers, binders) may include for example, acacia,albumin, alginic acid, calcium phosphate (dibasic), cellulose,carboxymethylcellulose, carboxymethylcellulose sodium,hydroxyethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, microcrystalline cellulose, dextran,dextrin, dextrates, sucrose, tylose, pregelatinized starch, calciumsulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose,disodium hydrogen phosphate, disodium phosphate, disodium pyrosulfite,polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose,compressible sugar, magnesium aluminum silicate, maltodextrin,polyethylene oxide, polymethacrylates, povidone, sodium alginate,tragacanth microcrystalline cellulose, starch, and zein. Exemplaryconcentrations of high molecular weight structural additives are from0.1% to 10% by weight. In other embodiments, a bulking agent (e.g.,mannitol, glycine) may be included.

Compositions may be suitable for parenteral administration. Exemplarycompositions are suitable for injection or infusion into an animal byany route available to the skilled worker, such as intraarticular,subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral(intraparenchymal), intracerebroventricular, intramuscular, intraocular,intraarterial, or intralesional routes. A parenteral formulationtypically may be a sterile, pyrogen-free, isotonic aqueous solution,optionally containing pharmaceutically acceptable preservatives.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringers'dextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present, suchas, for example, anti-microbials, anti-oxidants, chelating agents, inertgases and the like. See generally, Remington's Pharmaceutical Science,16th Ed., Mack Eds., 1980.

Pharmaceutical compositions described herein may be formulated forcontrolled or sustained delivery in a manner that provides localconcentration of the product (e.g., bolus, depot effect) and/orincreased stability or half-life in a particular local environment. Thecompositions can include the formulation of mTAs, PLCs, polypeptides,nucleic acids, or vectors disclosed herein with particulate preparationsof polymeric compounds such as polylactic acid, polyglycolic acid, etc.,as well as agents such as a biodegradable matrix, injectablemicrospheres, microcapsular particles, microcapsules, bioerodibleparticles beads, liposomes, and implantable delivery devices thatprovide for the controlled or sustained release of the active agentwhich then can be delivered as a depot injection. Techniques forformulating such sustained- or controlled-delivery means are known and avariety of polymers have been developed and used for the controlledrelease and delivery of drugs. Such polymers are typically biodegradableand biocompatible. Polymer hydrogels, including those formed bycomplexation of enantiomeric polymer or polypeptide segments, andhydrogels with temperature or pH sensitive properties, may be desirablefor providing drug depot effect because of the mild and aqueousconditions involved in trapping bioactive protein agents (e.g.,antibodies comprising an ultralong CDR3). See, for example, thedescription of controlled release porous polymeric microparticles forthe delivery of pharmaceutical compositions in WO 93/15722.

Suitable materials for this purpose include polylactides (see, e.g.,U.S. Pat. No. 3,773,919), polymers of poly-(a-hydroxycarboxylic acids),such as poly-D-(−)-3-hydroxybutyric acid (EP 133,988A), copolymers ofL-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers,22: 547-556 (1983)), poly(2-hydroxyethyl-methacrylate) (Langer et al.,J. Biomed. Mater. Res., 15: 167-277 (1981), and Langer, Chem. Tech., 12:98-105 (1982)), ethylene vinyl acetate, or poly-D(−)-3-hydroxybutyricacid. Other biodegradable polymers include poly(lactones),poly(acetals), poly(orthoesters), and poly(orthocarbonates).Sustained-release compositions also may include liposomes, which can beprepared by any of several methods known in the art (see, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82: 3688-92 (1985)). The carrieritself, or its degradation products, should be nontoxic in the targettissue and should not further aggravate the condition. This can bedetermined by routine screening in animal models of the target disorderor, if such models are unavailable, in normal animals.

Microencapsulation of recombinant proteins for sustained release hasbeen performed successfully with human growth hormone (rhGH),interferon-(rhIFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology. 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010. The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be depending on its molecular weight and composition. Lewis,“Controlled release of bioactive agents from lactide/glycolide polymer,”in: M. Chasin and R. Langer (Eds.), Biodegradable Polymers as DrugDelivery Systems (Marcel Dekker: New York, 1990), pp. 1-41. Additionalexamples of sustained release compositions include, for example, EP58,481A, U.S. Pat. No. 3,887,699, EP 158,277A, Canadian Patent No.1176565, U. Sidman et al., Biopolymers 22, 547 [1983], R. Langer et al.,Chem. Tech. 12, 98 [1982], Sinha et al., J. Control. Release 90, 261[2003], Zhu et al., Nat. Biotechnol. 18, 24 [2000], and Dai et al.,Colloids Surf B Biointerfaces 41, 117 [2005].

Bioadhesive polymers are also contemplated for use in or withcompositions of the present disclosure. Bioadhesives are synthetic andnaturally occurring materials able to adhere to biological substratesfor extended time periods. For example, Carbopol and polycarbophil areboth synthetic cross-linked derivatives of poly(acrylic acid).Bioadhesive delivery systems based on naturally occurring substancesinclude for example hyaluronic acid, also known as hyaluronan.Hyaluronic acid is a naturally occurring mucopolysaccharide consistingof residues of D-glucuronic and N-acetyl-D-glucosamine. Hyaluronic acidis found in the extracellular tissue matrix of vertebrates, including inconnective tissues, as well as in synovial fluid and in the vitreous andaqueous humor of the eye. Esterified derivatives of hyaluronic acid havebeen used to produce microspheres for use in delivery that arebiocompatible and biodegradable (see, for example, Cortivo et al.,Biomaterials (1991) 12:727-730; EP 517,565; WO 96/29998; Illum et al.,J. Controlled Rel. (1994) 29:133-141).

Both biodegradable and non-biodegradable polymeric matrices may be usedto deliver compositions of the present disclosure, and such polymericmatrices may comprise natural or synthetic polymers. Biodegradablematrices are preferred. The period of time over which release occurs isbased on selection of the polymer. Typically, release over a periodranging from between a few hours and three to twelve months is mostdesirable. Exemplary synthetic polymers which may be used to form thebiodegradable delivery system include: polymers of lactic acid andglycolic acid, polyamides, polycarbonates, polyalkylenes, polyalkyleneglycols, polyalkylene oxides, polyalkylene terepthalates, polyvinylalcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides,polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyanhydrides,polyurethanes and co-polymers thereof, poly(butic acid), poly(valericacid), alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers,cellulose esters, nitro celluloses, polymers of acrylic and methacrylicesters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose, celluloseacetate, cellulose propionate, cellulose acetate butyrate, celluloseacetate phthalate, carboxyethyl cellulose, cellulose triacetate,cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethylmethacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecylacrylate), polyethylene, polypropylene, poly(ethylene glycol),poly(ethylene oxide), poly(ethylene terephthalate), poly(vinylalcohols), polyvinyl acetate, poly vinyl chloride, polystyrene andpolyvinylpyrrolidone. Exemplary natural polymers include alginate andother polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion. The polymer optionally is in the formof a hydrogel (see, for example, WO 04/009664, WO 05/087201, Sawhney, etal., Macromolecules, 1993, 26, 581-587) that can absorb up to about 90%of its weight in water and further, optionally is cross-linked withmulti-valent ions or other polymers.

Delivery systems also include non-polymer systems that are lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-di- and tri-glycerides; hydrogelrelease systems; silastic systems; peptide based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which the product is contained in aform within a matrix such as those described in U.S. Pat. Nos.4,452,775, 4,675,189 and 5,736,152 and (b) diffusional systems in whicha product permeates at a controlled rate from a polymer such asdescribed in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.Liposomes containing the product may be prepared by methods knownmethods, such as for example (DE 3,218,121; Epstein et al., Proc. Natl.Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al., Proc. Natl. Acad.Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP143,949; EP 142,641; JP 83-118008; U.S. Pat. Nos. 4,485,045 and4,544,545; and EP 102,324).

Alternatively or additionally, the compositions may be administeredlocally via implantation into the affected area of a membrane, sponge,or other appropriate material on to which a PLC, mTA, nucleic acid, orvector disclosed herein has been absorbed or encapsulated. Where animplantation device is used, the device may be implanted into anysuitable tissue or organ, and delivery of an antibody comprising anultralong CDR3 antibody fragment, nucleic acid, or vector disclosedherein can be directly through the device via bolus, or via continuousadministration, or via catheter using continuous infusion.

A pharmaceutical composition comprising a PLC, mTA, nucleic acid, orvector disclosed herein may be formulated for inhalation, such as forexample, as a dry powder. Inhalation solutions also may be formulated ina liquefied propellant for aerosol delivery. In yet another formulation,solutions may be nebulized. Additional pharmaceutical composition forpulmonary administration include, those described, for example, in WO94/20069, which discloses pulmonary delivery of chemically modifiedproteins. For pulmonary delivery, the particle size should be suitablefor delivery to the distal lung. For example, the particle size may befrom 1 μm to 5 μm; however, larger particles may be used, for example,if each particle is fairly porous.

Certain formulations containing antibodies comprising a PLC, mTA,nucleic acid, or vector disclosed herein may be administered orally.Formulations administered in this fashion may be formulated with orwithout those carriers customarily used in the compounding of soliddosage forms such as tablets and capsules. For example, a capsule can bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. Additional agents may be includedto facilitate absorption of a selective binding agent. Diluents,flavorings, low melting point waxes, vegetable oils, lubricants,suspending agents, tablet disintegrating agents, and binders also can beemployed.

Another preparation may involve an effective quantity of an antibodycomprising a PLC, mTA, nucleic acid, or vector disclosed herein in amixture with non-toxic excipients which are suitable for the manufactureof tablets. By dissolving the tablets in sterile water, or anotherappropriate vehicle, solutions may be prepared in unit dose form.Suitable excipients include, but are not limited to, inert diluents,such as calcium carbonate, sodium carbonate or bicarbonate, lactose, orcalcium phosphate; or binding agents, such as starch, gelatin, oracacia; or lubricating agents such as magnesium stearate, stearic acid,or talc.

Suitable and/or preferred pharmaceutical formulations may be determinedin view of the present disclosure and general knowledge of formulationtechnology, depending upon the intended route of administration,delivery format, and desired dosage. Regardless of the manner ofadministration, an effective dose may be calculated according to patientbody weight, body surface area, or organ size. Further refinement of thecalculations for determining the appropriate dosage for treatmentinvolving each of the formulations described herein are routinely madein the art and is within the ambit of tasks routinely performed in theart. Appropriate dosages may be ascertained through use of appropriatedose-response data.

Vectors, Host Cells and Recombinant Methods

A TA, as disclosed herein, may be expressed by recombinant methods.Generally, a nucleic acid encoding a TA may be isolated and insertedinto a replicable vector for further cloning (amplification of the DNA)or for expression. DNA encoding the TA may be prepared by PCRamplification and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically tonucleotides encoding a TA). In an exemplary embodiment, nucleic acidencoding a TA is PCR amplified, restriction enzyme digested and gelpurified. The digested DNA may be inserted into a replicable vector. Thereplicable vector containing the digested DNA insertion may betransformed or transduced into a host cell for further cloning(amplification of the DNA) or for expression. Host cells may beprokaryotic or eukaryotic cells.

Polynucleotide sequences encoding polypeptide components of the mTAs orPLCs disclosed herein may be obtained by PCR amplification withoverlapping oligonucleotide primers. Polynucleotide sequences may beisolated and sequenced from TA producing cells. Alternatively,polynucleotides may be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic and/or eukaryotic hosts.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism may be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

TAs may be expressed in intracellularly (e.g., cytoplasm) orextracellularly (e.g., secretion). For extracellular expression, thevector may comprise a secretion signal which enables translocation ofthe TA to the outside of the cell.

Suitable host cells for cloning or expression of TA-encoding vectorsinclude prokaryotic or eukaryotic cells. The host cell may be aeukaryotic. Examples of eukaryotic cells include, but are not limitedto, Human Embryonic Kidney (HEK) cell, Chinese Hamster Ovary (CHO) cell,fungi, yeasts, invertebrate cells (e.g., plant cells and insect cells),lymphoid cell (e.g., YO, NSO, Sp20 cell). Other examples of suitablemammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7); baby hamster kidney cells (BHK); mouse Sertoli cells; monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells(HepG2); mouse mammary tumor (MMT 060562); TR1 cells; MRC 5 cells; andFS4 cells. The host cell may be a prokaryotic cell (e.g., E. coli).

Host cells may be transformed with vectors containing nucleotidesencoding a TA. Transformed host cells may be cultured in media. Themedia may be supplemented with one or more agents for inducingpromoters, selecting transformants, or amplifying or expressing thegenes encoding the desired sequences. Methods for transforming hostcells are known in the art and may include electroporation, calciumchloride, or polyethylene glycol/DMSO.

Alternatively, host cells may be transfected or transduced with vectorscontaining nucleotides encoding a TA. Transfected or transduced hostcells may be cultured in media. The media may be supplemented with oneor more agents for inducing promoters, selecting transfected ortransduced cells, or expressing genes encoding the desired sequences.

The expressed TAs may be secreted into and recovered from the periplasmof the host cells or transported into the culture media. Proteinrecovery from the periplasm may involve disrupting the host cell.Disruption of the host cell may comprise osmotic shock, sonication orlysis. Centrifugation or filtration may be used to remove cell debris orwhole cells. The TAs may be further purified, for example, by affinityresin chromatography.

Alternatively, TAs that are secreted into the culture media may beisolated therein. Cells may be removed from the culture and the culturesupernatant being filtered and concentrated for further purification ofthe proteins produced. The expressed polypeptides can be furtherisolated and identified using commonly known methods such aspolyacrylamide gel electrophoresis (PAGE) and Western blot assay.

TA production may be conducted in large quantity by a fermentationprocess. Various large-scale fed-batch fermentation procedures areavailable for production of recombinant proteins. Large-scalefermentations have at least 1000 liters of capacity, preferably about1,000 to 100,000 liters of capacity. These fermentors use agitatorimpellers to distribute oxygen and nutrients, especially glucose (apreferred carbon/energy source). Small scale fermentation refersgenerally to fermentation in a fermentor that is no more thanapproximately 100 liters in volumetric capacity, and can range fromabout 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD550 of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the TAs disclosed herein,various fermentation conditions can be modified. For example, to improvethe proper assembly and folding of the secreted TA polypeptides,additional vectors overexpressing chaperone proteins, such as Dsbproteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolylcis,trans-isomerase with chaperone activity) may be used to co-transformthe host prokaryotic cells. The chaperone proteins have beendemonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the presentdisclosure. For example, host cell strains may be modified to effectgenetic mutation(s) in the genes encoding known bacterial proteases suchas Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available.

Standard protein purification methods known in the art can be employed.The following procedures are exemplary of suitable purificationprocedures: fractionation on immunoaffinity or ion-exchange columns,ethanol precipitation, reverse phase HPLC, chromatography on silica oron a cation-exchange resin such as DEAE, chromatofocusing, SDS-PAGE,ammonium sulfate precipitation, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography and gelfiltration using, for example, Sephadex G-75.

TAs may be concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pellicon®ultrafiltration unit.

Protease inhibitors or protease inhibitor cocktails may be included inany of the foregoing steps to inhibit proteolysis of the TA.

In some cases, a TA or fragment thereof may not be biologically activeupon isolation. Various methods for “refolding” or converting apolypeptide to its tertiary structure and generating disulfide linkages,can be used to restore biological activity. Such methods includeexposing the solubilized polypeptide to a pH usually above 7 and in thepresence of a particular concentration of a chaotrope. The selection ofchaotrope is very similar to the choices used for inclusion bodysolubilization, but usually the chaotrope is used at a lowerconcentration and is not necessarily the same as chaotropes used for thesolubilization. In most cases the refolding/oxidation solution will alsocontain a reducing agent or the reducing agent plus its oxidized form ina specific ratio to generate a particular redox potential allowing fordisulfide shuffling to occur in the formation of the protein's cysteinebridge(s). Some of the commonly used redox couples includecystein/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride,dithiothreitol(DTT)/dithiane DTT, and2-mercaptoethanol(bME)/di-thio-b(ME). In many instances, a cosolvent maybe used to increase the efficiency of the refolding, and common reagentsused for this purpose include glycerol, polyethylene glycol of variousmolecular weights, arginine and the like.

Kits/Articles of Manufacture

As an additional aspect, the present disclosure includes kits whichcomprise one or more compounds or compositions packaged in a mannerwhich facilitates their use to practice methods of the presentdisclosure. In one embodiment, such a kit includes a compound orcomposition described herein (e.g. a PLC or mTA alone or in combinationwith a second agent), packaged in a container with a label affixed tothe container or a package insert that describes use of the compound orcomposition in practicing the method. Suitable containers include, forexample, bottles, vials, syringes, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The container mayhave a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). The article of manufacture may comprise(a) a first container with a composition contained therein, wherein thecomposition comprises a PLC or mTA as disclosed herein; and (b) a secondcontainer with a composition contained therein, wherein the compositioncomprises a further therapeutic agent. The article of manufacture inthis embodiment disclosed herein may further comprise a package insertindicating that the first and second compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes. Preferably, the compound orcomposition is packaged in a unit dosage form. The kit may furtherinclude a device suitable for administering the composition according toa specific route of administration or for practicing a screening assay.Preferably, the kit contains a label that describes use of the PLC ormTA composition.

In certain embodiments, the composition comprising the antibody isformulated in accordance with routine procedures as a pharmaceuticalcomposition adapted for intravenous administration to mammals, such ashumans, bovines, felines, canines, and murines. Typically, compositionsfor intravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilisingagent and a local anaesthetic such as lignocaine to ease pain at thesite of the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The amount of the composition described herein which will be effectivein the treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a Therapeuticprotein can be determined by standard clinical techniques. In addition,in vitro assays may optionally be employed to help identify optimaldosage ranges. The precise dose to be employed in the formulation willalso depend on the route of administration, and the seriousness of thedisease or disorder, and should be decided according to the judgment ofthe practitioner and each patient's circumstances. Effective doses areextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

Definitions

The terms below, as used herein, have the following meanings, unlessindicated otherwise:

“Amino” refers to the —NH₂ radical.

“Hydroxy” or “hydroxyl” refers to the —OH radical.

“Nitro” refers to the —NO₂ radical.

“Oxo” refers to the ═O substituent.

“Oxime” refers to the ═N—OH substituent.

“Alkyl” refers to a straight or branched hydrocarbon chain radical, hasfrom one to thirty carbon atoms, and is attached to the rest of themolecule by a single bond. Alkyls comprising any number of carbon atomsfrom 1 to 30 are included. An alkyl comprising up to 30 carbon atoms isreferred to as a C₁-C₃₀ alkyl, likewise, for example, an alkylcomprising up to 12 carbon atoms is a C₁-C₁₂ alkyl. Alkyls (and othermoieties defined herein) comprising other numbers of carbon atoms arerepresented similarly. Alkyl groups include, but are not limited to,C₁-C₃₀ alkyl, C₁-C₂₀ alkyl, C₁-C₁₅ alkyl, C₁-C₁₀ alkyl, C₁-C₈ alkyl,C₁-C₆ alkyl, C₁-C₄ alkyl, C₁-C₃ alkyl, C₁-C₂ alkyl, C₂-C₈ alkyl, C₃-C₈alkyl and C₄-C₈ alkyl. Representative alkyl groups include, but are notlimited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl),n-butyl, i-butyl, s-butyl, n-pentyl, 1,1-dimethylethyl (t-butyl),3-methylhexyl, 2-methylhexyl, vinyl, allyl, propynyl, and the like.Alkyl comprising unsaturations include alkenyl and alkynyl groups.Unless stated otherwise specifically in the specification, an alkylgroup may be optionally substituted as described below.

“Alkylene” or “alkylene chain” refers to a straight or branched divalenthydrocarbon chain, as described for alkyl above. Unless stated otherwisespecifically in the specification, an alkylene group may be optionallysubstituted as described below.

“Alkoxy” refers to a radical of the formula —OR_(a) where R_(a) is analkyl radical as defined. Unless stated otherwise specifically in thespecification, an alkoxy group may be optionally substituted asdescribed below.

“Aryl” refers to a radical derived from a hydrocarbon ring systemcomprising hydrogen, 6 to 30 carbon atoms and at least one aromaticring. The aryl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems. Aryl radicals include, but are not limited to, aryl radicalsderived from the hydrocarbon ring systems of aceanthrylene,acenaphthylene, acephenanthrylene, anthracene, azulene, benzene,chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane,indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene, andtriphenylene. Unless stated otherwise specifically in the specification,the term “aryl” or the prefix “ar-” (such as in “aralkyl”) is meant toinclude aryl radicals that are optionally substituted.

“Cycloalkyl” or “carbocycle” refers to a stable, non-aromatic,monocyclic or polycyclic carbocyclic ring, which may include fused orbridged ring systems, which is saturated or unsaturated. Representativecycloalkyls or carbocycles include, but are not limited to, cycloalkylshaving from three to fifteen carbon atoms, from three to ten carbonatoms, from three to eight carbon atoms, from three to six carbon atoms,from three to five carbon atoms, or three to four carbon atoms.Monocyclic cycloalkyls or carbocycles include, for example, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.Polycyclic cycloalkyls or carbocycles include, for example, adamantyl,norbornyl, decalinyl, bicyclo[3.3.0]octane, bicyclo[4.3.0]nonane,cis-decalin, trans-decalin, bicyclo[2.1.1]hexane, bicyclo[2.2.1]heptane,bicyclo[2.2.2]octane, bicyclo[3.2.2]nonane, and bicyclo[3.3.2]decane,and 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Unless otherwise statedspecifically in the specification, a cycloalkyl or carbocycle group maybe optionally substituted. Illustrative examples of cycloalkyl groupsinclude, but are not limited to, the following moieties:

and the like.

“Fused” refers to any ring structure described herein which is fused toan existing ring structure. When the fused ring is a heterocyclyl ringor a heteroaryl ring, any carbon atom on the existing ring structurewhich becomes part of the fused heterocyclyl ring or the fusedheteroaryl ring may be replaced with a nitrogen atom.

“Halo” or “halogen” refers to bromo, chloro, fluoro or iodo.

“Haloalkyl” refers to an alkyl radical, as defined above, that issubstituted by one or more halo radicals, as defined above, e.g.,trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl,2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl,1,2-dibromoethyl, and the like. Unless stated otherwise specifically inthe specification, a haloalkyl group may be optionally substituted.

“Haloalkoxy” similarly refers to a radical of the formula —OR_(a) whereR_(a) is a haloalkyl radical as defined. Unless stated otherwisespecifically in the specification, a haloalkoxy group may be optionallysubstituted as described below.

“Heterocycloalkyl” or “heterocyclyl” or “heterocyclic ring” or“heterocycle” refers to a stable 3- to 24-membered non-aromatic ringradical comprising 2 to 23 carbon atoms and from one to 8 heteroatomsselected from the group consisting of nitrogen, oxygen, phosphorous andsulfur. Unless stated otherwise specifically in the specification, theheterocyclyl radical may be a monocyclic, bicyclic, tricyclic ortetracyclic ring system, which may include fused or bridged ringsystems; and the nitrogen, carbon or sulfur atoms in the heterocyclylradical may be optionally oxidized; the nitrogen atom may be optionallyquaternized; and the heterocyclyl radical may be partially or fullysaturated. Examples of such heterocyclyl radicals include, but are notlimited to, azetidinyl, dioxolanyl, thienyl[1,3]dithianyl,decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl,isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl,piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl,quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl,tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl,1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl,12-crown-4,15-crown-5,18-crown-6,21-crown-7, aza-18-crown-6,diaza-18-crown-6, aza-21-crown-7, and diaza-21-crown-7. Unless statedotherwise specifically in the specification, a heterocyclyl group may beoptionally substituted. Illustrative examples of heterocycloalkylgroups, also referred to as non-aromatic heterocycles, include:

and the like. The term heterocycloalkyl also includes all ring forms ofthe carbohydrates, including but not limited to the monosaccharides, thedisaccharides and the oligosaccharides. Unless otherwise noted,heterocycloalkyls have from 2 to 10 carbons in the ring. It isunderstood that when referring to the number of carbon atoms in aheterocycloalkyl, the number of carbon atoms in the heterocycloalkyl isnot the same as the total number of atoms (including the heteroatoms)that make up the heterocycloalkyl (i.e. skeletal atoms of theheterocycloalkyl ring). Unless stated otherwise specifically in thespecification, a heterocycloalkyl group may be optionally substituted.

The term “heteroaryl” as used herein, alone or in combination, refers tooptionally substituted aromatic monoradicals containing from about fiveto about twenty skeletal ring atoms, where one or more of the ring atomsis a heteroatom independently selected from among oxygen, nitrogen,sulfur, phosphorous, silicon, selenium and tin but not limited to theseatoms and with the proviso that the ring of said group does not containtwo adjacent O or S atoms. In embodiments in which two or moreheteroatoms are present in the ring, the two or more heteroatoms can bethe same as each another, or some or all of the two or more heteroatomscan each be different from the others. The term heteroaryl includesoptionally substituted fused and non-fused heteroaryl radicals having atleast one heteroatom. The term heteroaryl also includes fused andnon-fused heteroaryls having from five to about twelve skeletal ringatoms, as well as those having from five to about ten skeletal ringatoms. Bonding to a heteroaryl group can be via a carbon atom or aheteroatom. Thus, as a non-limiting example, an imidiazole group may beattached to a parent molecule via any of its carbon atoms(imidazol-2-yl, imidazol-4-yl or imidazol-5-yl), or its nitrogen atoms(imidazol-1-yl or imidazol-3-yl). Likewise, a heteroaryl group may befurther substituted via any or all of its carbon atoms, and/or any orall of its heteroatoms. A fused heteroaryl radical may contain from twoto four fused rings where the ring of attachment is a heteroaromaticring and the other individual rings may be alicyclic, heterocyclic,aromatic, heteroaromatic or any combination thereof. A non-limitingexample of a single ring heteroaryl group includes pyridyl; fused ringheteroaryl groups include benzimidazolyl, quinolinyl, acridinyl; and anon-fused bi-heteroaryl group includes bipyridinyl. Further examples ofheteroaryls include, without limitation, furanyl, thienyl, oxazolyl,acridinyl, azepinyl, phenazinyl, benzimidazolyl, benzindolyl,benzofuranyl, benzofuranonyl, benzoxazolyl, benzothiazolyl,benzothiadiazolyl, benzothiophenyl, benzoxadiazolyl, benzodioxolyl,benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl,benzotriazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl,benzopyranonyl, benzothienyl (benzothiophenyl),benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl,dibenzofuranyl, dibenzothiophenyl, furanonyl, imidazolyl, indolyl,isoxazolyl, isoquinolinyl, indolizinyl, indazolyl, isoindolyl,indolinyl, isoindolinyl, indolizinyl, isothiazolyl,isoindolyloxadiazolyl, indazolyl, naphthyridinyl, oxadiazolyl,2-oxoazepinyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl,1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl,phenothiazinyl, phenoxazinyl, pyridinyl, pyridazinyl, pyrimidinyl,pyrazinyl, pyrrolyl, pyrazinyl, pyrazolyl, purinyl, phthalazinyl,pteridinyl, quinolinyl, quinazolinyl, quinoxalinyl, quinuclidinyl,triazolyl, tetrazolyl, thiazolyl, triazinyl, thiadiazolyl,tetrahydroquinolinyl, thiazolyl, and thiophenyl and the like, and theiroxides, such as for example pyridyl-N-oxide. Illustrative examples ofheteroaryl groups include the following moieties:

and the like.

All the above groups may be either substituted or unsubstituted. Theterm “substituted” as used herein means any of the above groups (e.g.,alkyl, alkylene, alkoxy, aryl, cycloalkyl, haloalkyl, heterocyclyland/or heteroaryl) may be further functionalized wherein at least onehydrogen atom is replaced by a bond to a non-hydrogen atom substituent.Unless stated specifically in the specification, a substituted group mayinclude one or more substituents selected from: oxo, amino, —CO₂H,nitrile, nitro, hydroxyl, thiooxy, alkyl, alkylene, alkoxy, aryl,cycloalkyl, heterocyclyl, heteroaryl, dialkylamines, arylamines,alkylarylamines, diarylamines, trialkylammonium (—N⁺R₃), N-oxides,imides, and enamines; a silicon atom in groups such as trialkylsilylgroups, dialkylarylsilyl groups, alkyldiarylsilyl groups, triarylsilylgroups, perfluoroalkyl or perfluoroalkoxy, for example, trifluoromethylor trifluoromethoxy. “Substituted” also means any of the above groups inwhich one or more hydrogen atoms are replaced by a higher-order bond(e.g., a double- or triple-bond) to a heteroatom such as oxygen in oxo,carbonyl, carboxyl, and ester groups; and nitrogen in groups such asimines, oximes, hydrazones, and nitriles. For example, “substituted”includes any of the above groups in which one or more hydrogen atoms arereplaced with —NH₂, —NR_(g)C(═O)NR_(g)R_(h), —NR_(g)C(═O)OR_(h),—NR_(g)SO₂R_(h), —OC(═O)NR_(g)R_(h), —OR_(g), —SR_(g), —SOR_(g),—SO₂R_(g), —OSO₂R_(g), —SO₂OR_(g), ═NSO₂R_(g), and —SO₂NR_(g)R_(h). Inthe foregoing, R_(g) and R_(h) are the same or different andindependently hydrogen, alkyl, alkoxy, alkylamino, thioalkyl, aryl,aralkyl, cycloalkyl, cycloalkylalkyl, haloalkyl, heterocyclyl,N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/orheteroarylalkyl. In addition, each of the foregoing substituents mayalso be optionally substituted with one or more of the abovesubstituents. Furthermore, any of the above groups may be substituted toinclude one or more internal oxygen, sulfur, or nitrogen atoms. Forexample, an alkyl group may be substituted with one or more internaloxygen atoms to form an ether or polyether group. Similarly, an alkylgroup may be substituted with one or more internal sulfur atoms to forma thioether, disulfide, etc.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl” as defined above.Further, an optionally substituted group may be un-substituted (e.g.,—CH₂CH₃), fully substituted (e.g., —CF₂CF₃), mono-substituted (e.g.,—CH₂CH₂F) or substituted at a level anywhere in-between fullysubstituted and mono-substituted (e.g., —CH₂CHF₂, —CH₂CF₃, —CF₂CH₃,—CFHCHF₂, etc). It will be understood by those skilled in the art withrespect to any group containing one or more substituents that suchgroups are not intended to introduce any substitution or substitutionpatterns (e.g., substituted alkyl includes optionally substitutedcycloalkyl groups, which in turn are defined as including optionallysubstituted alkyl groups, potentially ad infinitum) that are stericallyimpractical and/or synthetically non-feasible. Thus, any substituentsdescribed should generally be understood as having a maximum molecularweight of about 1,000 daltons, and more typically, up to about 500daltons.

A “tautomer” refers to a proton shift from one atom of a molecule toanother atom of the same molecule. The compounds presented herein mayexist as tautomers. Tautomers are compounds that are interconvertible bymigration of a hydrogen atom, accompanied by a switch of a single bondand adjacent double bond. In bonding arrangements where tautomerizationis possible, a chemical equilibrium of the tautomers will exist. Alltautomeric forms of the compounds disclosed herein are contemplated. Theexact ratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Some examples of tautomericinterconversions include:

A “metabolite” of a compound disclosed herein is a derivative of thatcompound that is formed when the compound is metabolized. The term“active metabolite” refers to a biologically active derivative of acompound that is formed when the compound is metabolized. The term“metabolized,” as used herein, refers to the sum of the processes(including, but not limited to, hydrolysis reactions and reactionscatalyzed by enzymes, such as, oxidation reactions) by which aparticular substance is changed by an organism. Thus, enzymes mayproduce specific structural alterations to a compound. For example,cytochrome P450 catalyzes a variety of oxidative and reductive reactionswhile uridine diphosphate glucuronyl transferases catalyze the transferof an activated glucuronic-acid molecule to aromatic alcohols, aliphaticalcohols, carboxylic acids, amines and free sulfhydryl groups. Furtherinformation on metabolism may be obtained from The Pharmacological Basisof Therapeutics, 9th Edition, McGraw-Hill (1996). Metabolites of thecompounds disclosed herein can be identified either by administration ofcompounds to a host and analysis of tissue samples from the host, or byincubation of compounds with hepatic cells in vitro and analysis of theresulting compounds. Both methods are well known in the art. Metabolitesof a compound may be formed by oxidative processes and correspond to thecorresponding hydroxy-containing compound. A compound may be metabolizedto one or more pharmacologically active metabolites.

As used herein, a derivative of a peptide refers to, but is not limitedto, a modified peptide that allows for staple and/or HEM attachment(such as one or more amino acid residue replacements or L- vs D-aminoacid replacements), a fragment, an analog with one or more additionalamino acids, a complex and/or an aggregate of the peptide. A derivativeof a peptide may be a homolog that has at least 50% homology withrespect to the peptide. A derivative of a peptide may be a homolog thathas at least 60% homology with respect to the peptide. A derivative of apeptide may be a homolog that has at least 70% homology with respect tothe peptide. A derivative of a peptide may be a homolog that has atleast 80% homology with respect to the peptide. A derivative of apeptide may be a homolog that has at least 90% homology with respect tothe peptide.

“Pharmaceutically acceptable” refers to approved or approvable by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, including humans.

“Pharmaceutically acceptable salt” refers to a salt of a compound thatis pharmaceutically acceptable and that possesses the desiredpharmacological activity of the parent compound.

“Pharmaceutically acceptable excipient, carrier or adjuvant” refers toan excipient, carrier or adjuvant that may be administered to a subject,together with at least one antibody of the present disclosure, and whichdoes not destroy the pharmacological activity thereof and is nontoxicwhen administered in doses sufficient to deliver a therapeutic amount ofthe compound.

“Pharmaceutically acceptable vehicle” refers to a diluent, adjuvant,excipient, or carrier with which at least one antibody of the presentdisclosure is administered.

Terms such as “treating” or “treatment” or “to treat” or “alleviating”or “to alleviate” may refer to: 1) therapeutic measures that cure, slowdown, lessen symptoms of, and/or halt progression of a diagnosedpathologic condition or disorder; and/or 2) prophylactic or preventativemeasures that prevent and/or slow the development of a targetedpathologic condition or disorder. “Treatment” refers to clinicalintervention in an attempt to alter the natural course of the individualor cell being treated, and can be performed either for prophylaxis orduring the course of clinical pathology. Desirable effects of treatmentinclude preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. Thus those in need of treatment mayinclude those already with the disorder; those prone to have thedisorder; and those in whom the disorder is to be prevented.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that functionsimilarly to the naturally occurring amino acids. Naturally occurringamino acids are those encoded by the genetic code, as well as thoseamino acids that are later modified, e.g., hydroxyproline,gamma-carboxyglutamate, and O-phosphoserine. Amino acid analogs refersto compounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an alpha carbon that is bound to a hydrogen,a carboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs can have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

As used herein, the term “therapeutic agent” or “peptide therapeuticagent” or “therapeutic peptide” refers to a protein or peptide thatmodulates the activity of another protein, peptide, cell or tissue.Modulating the activity can comprise increasing, decreasing,stimulating, or preventing the activity or expression of the protein,peptide, cell or tissue. Therapeutic agents may modulate the activity ofproteins or peptides involved in the etiology of a disease or disorder.Exemplary TAs may include, but are not limited to, at least a portion ofa hormone, kinase, receptor, ligand, growth factor, regulatory protein,metabolic protein, cytokine, chemokine, interferon, phosphatase,antibody or any combination thereof.

“Disorder” or “disease” refers to a condition that would benefit fromtreatment with a substance/molecule (e.g., a mTA or PLC as disclosedherein) or method disclosed herein. This includes chronic and acutedisorders or diseases including those pathological conditions whichpredispose the mammal to the disorder in question.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, rodents (e.g., mice and rats), and monkeys;domestic and farm animals; and zoo, sports, laboratory, or pet animals,such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, etc. Insome embodiments, the mammal is selected from a human, rodent, ormonkey.

EXAMPLES Example 1. Synthesis of Exemplary Lipid Derivatives (Scheme A)

tert-Butyl (2-(2-(2-tetradecanamidoethoxy)ethoxy)ethyl)carbamate(A-2-a).

N-t-Boc-amido-dPEG3-amine (0.5 g, 2.0 mmol) was added to a solution ofmyristic acid (0.46 g, 2.0 mmol) in 10 ml of dry DMF, followed by HATU(0.8 g, 2.1 mmol) and DIEA (0.45 mL, 2.4 mmol). The mixture was stirredat RT for 6 h and the solvent was evaporated in vacuo. The crudematerial was dissolved in EtOAc, washed with cold 1% HCl, saturatedNaHCO₃ and brine, dried over Na₂SO₄, filtered and concentrated. Thecrude material was purified by flash column chromatography on silica gelwith a gradient 25-50% EtOAc in hexanes to afford 0.83 g of desiredcompound as a white solid (Yield 90%). m/z (ESI+) 459.6 (M+H).

tert-Butyl(2-(2-(2-((4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenamido)ethoxy)ethoxy)ethyl)carbamate(A-2-b).

The title compound was prepared using analogous conditions as theprocedure for A-2-a, using N-t-Boc-amido-dPEG3-amine (0.25 g, 1.0 mmol),docosahexaenoic acid (0.33 g, 1.0 mmol), HATU (0.41 g, 1.1 mmol) andDIEA (0.22 mL, 1.2 mmol in dry DMF (5 mL). Yield 68%, brown oil. ¹H NMR(500 MHz; CDCl₃): δ 0.97 (t, J=6.0 Hz, 3H), 1.44 (s, 9H), 2.07 (t, J=5.0Hz, 2H), 2.25 (J=5.0 Hz, 2H), 2.41 (dd, J=5.0, 6.5 Hz, 2H), 2.79-2.87(m, 10H), 3.32 (t, J=5.5 Hz, 2H), 3.45 (dd, J=5.0, 6.0 Hz, 2H), 3.55 (t,J=5.0 Hz, 4H), 3.59-3.62 (m, 4H), 4.97 (s, 1H), 5.30-5.42 (m, 12H), 6.03(s, 1H).

tert-Butyl(2-(2-(2-((R)-4-((3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamido)ethoxy)ethoxy)ethyl)carbamate (A-2-c).

N-t-Boc-amido-dPEG3-amine (0.14 g, 0.55 mmol) was added to a solution ofNHS-activated lithocholic acid ester (0.24 g, 0.5 mmol) in 5 ml of dryDMF, followed by DIEA (0.18 mL, 1.0 mmol). The mixture was stirred at RTfor 16 h, and the solvent was evaporated in vacuo. The crude material isdissolved in DCM, washed with cold 1% HCl, saturated NaHCO₃ and brine,dried over Na₂SO₄, filtered and concentrated. The crude material waspurified by flash column chromatography on silica gel with a gradient1-5% methanol in DCM to afford 0.48 g of desired compound as a whitesolid (Yield 80%). ¹H NMR (500 MHz; CDCl₃): δ 0.62 (s, 3H), 0.90-2.25(m, 30H), 3.31 (s, 2H), 3.43-3.46 (s, 2H), 3.52-3.56 (m, 4H), 3.59-3.60(m, 6H), 4.94 (s, 1H), 6.05 (s, 1H); m/z (ESI+) 628.6 (M+H). m/z (ESI+)607.5 (M+H).

N-(2-(2-(2-(2-Bromoacetamido)ethoxy)ethoxy)ethyl)tetradecanamide (A-3-a)

TFA (2 ml, 26 mmol) was added to a solution of A-2-a (0.46 g, 1 mmol) in10 ml of DCM, and the mixture was stirred at RT for 3 h. The reactionmixture was concentrated, and the crude material was lyophilized toobtain a colorless oil that was dissolved in 10 ml of DCM. Bromoaceticanhydride (0.31 g, 1.2 mmol) was added, followed by DIEA (0.52 ml, 2.5mmol), and the mixture was stirred at RT for 3 h. The reaction mixturewas extracted with DCM and EtOAc, washed with 1% HCl, saturated NaHCO₃and brine, dried over Na₂SO₄, filtered and concentrated. The crudematerial was purified by flash column chromatography on silica gel witha gradient 20-50% EtOAc in petroleum ether with 5% methanol to obtain0.37 g of desired compound as a white solid (combined yield over twosteps, 78%). ¹H NMR (500 MHz; CDCl₃): δ 0.88 (t, J=6.5 Hz, 3H),1.25-1.32 (m, 20H), 1.62 (t, J=7.5 Hz, 2H), 2.18 (t, J=8.0 Hz, 2H), 3.47(dd, J=5.0, 10.0 Hz, 2H), 3.50 (dd, J=5.0, 10.0 Hz, 2H), 3.56-3.58 (m,2H), 3.59-3.62 (m, 2H), 3.63 (d, J=5.5 Hz, 5H), 3.88 (s, 2H), 5.92-5.93(m, 1H), 6.94 (s, 1H); 7.04-7.17 (m, 1H); 13C NMR (100 MHz; CDCl3): δ14.55, 23.12, 26.20, 29.76, 29.79, 29.95, 30.06, 30.08, 30.09, 30.11,32.35, 37.23, 69.84, 70.46, 70.83 (2), 166.06, 173.84; m/z (ESI+) 480.6(M+H).

(4Z,7Z,10Z,13Z,16Z,19Z)—N-(2-(2-(2-(2-bromoacetamido)ethoxy)ethoxy)ethyl)docosa-4,7,10,13,16,19-hexaenamide (A-3-b)

The title compound was prepared using analogous conditions as theprocedure for A-3-a. Yield 67%, brown oil. ¹H NMR (500 MHz; CDCl₃): δ0.96 (t, J=5.0 Hz, 3H), 2.07 (q, J=7.6 Hz, 2H), 2.23 (J=7.2 Hz, 2H),2.40 (dd, J=7.2, 13.8 Hz, 2H), 2.79-2.84 (m, 1OH), 3.44-3.51 (m, 4H),3.55-3.62 (m, 4H), 3.63 (s, 4H), 3.88 (s, 2H), 5.78-5.42 (m, 12H), 5.94(s, 1H), 6.92 (s, 1H); m/z (ESI+) 559.8 (M+H).

(R)—N-(2-(2-(2-(2-bromoacetamido)ethoxy)ethoxy)ethyl)-4-((3R,5R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanamide(A-3-c)

The title compound was prepared using analogous conditions as theprocedure for A-3-a, replacing A-2-a with A-2-c. Yield 87%, white solid.¹H NMR (500 MHz; CDCl₃): δ 0.83 (t, J=4.0 Hz, 3H), 0.85-0.94 (m, 6H),1.00-1.91 (m, 28H), 3.41-3.64 (m, 12H), 3.85-3.88 (m, 2H), 4.86-4.94 (m,1H), 6.05 (t, J=7.2 Hz, 1H), 7.04 (s, 1H); m/z (ESI+) 628.6 (M+H).

Example 2. Synthesis of Exemplary Lipid Derivatives (Schemes B-1 andB-2)

A solution of 2-(2-(2-aminoethoxy)ethoxy)ethanol (100 mg, 0.671 mmol, 89μL) in 4.8 mL of acetonitrile/water (6:1) was treated with di-tert-butyldicarbonate (151 mg, 0.691 mmol), followed by 0.5 mL of 1 N NaOH (aq).After stirring at RT for 45 min, the organic solvent was removed invacuo, the residue was dissolved in saturated NH₄Cl (aq), and thedesired carbamate was extracted with EtOAc. Removal of EtOAc provided 57mg (34% yield) of tert-butyl 2-(2-(2-hydroxyethoxy)ethoxy)ethylcarbamateas a colorless oil.

A solution of tert-butyl 2-(2-(2-hydroxyethoxy)ethoxy)ethylcarbamate(68.5 mg, 0.275 mmol) and myristyl tosylate (101 mg, 0.275 mmol) in 1.4mL of t-amyl alcohol was treated with potassium tert-butoxide (61.6 mg,0.550 mmol) and potassium iodide (4.6 mg, 0.028 mmol). After heating to90° C. for 2 h, the reaction was allowed to cool to rt, quenched withsaturated NH₄Cl (aq), then extracted with EtOAc and dried over Na₂SO₄.Concentration and subsequent purification via flash columnchromatography on silica gel afforded 44 mg (36% yield) of tert-butyl2-(2-(2-(tetradecyloxy)ethoxy)ethoxy)ethylcarbamate as a colorless oil.

A solution of tert-butyl2-(2-(2-(tetradecyloxy)ethoxy)ethoxy)ethylcarbamate (26 mg, 0.058 mmol)in 1.2 mL of DCM was treated with 0.29 mL of trifluoroacetic acid. Afterstirring at rt for 50 min, the mixture was concentrated and re-dissolvedin 2 mL DCM. A 1-mL aliquot of this solution was taken into a separatevial, which was cooled to 0° C. and subsequently charged withbromoacetic anhydride (10.3 mg, 0.040 mmol) and DIEA (11 μL, 0.063mmol). After stirring at rt for 12 h, the reaction mixture was purifiedvia flash column chromatography to provide 7.5 mg (55% yield over 2steps) of2-bromo-N-(2-(2-(2-(tetradecyloxy)ethoxy)ethoxy)-ethyl)acetamide as anoff-white solid.

tert-Butyl 2-(2-(2-(2-bromoethoxy)ethoxy)ethoxy)ethylcarbamate (B-2-a)

A solution of 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol (400 mg, 2.07mmol, 0.33 mL) in 15 mL of acetonitrile/water (6:1) was treated withdi-tert-butyl dicarbonate (603 mg, 2.77 mmol), followed by 2.8 mL of 1 NNaOH (aq). After stirring at RT for 45 min, the organic solvent wasremoved in vacuo, the residue was dissolved in saturated NH₄Cl (aq), andthe desired carbamate was extracted with EtOAc. Removal of EtOAcprovided the crude carbamate as a colorless oil.

This oil was dissolved in DCM and treated with p-toluenesulfonylchloride (1.18 g, 6.21 mmol) and pyridine (0.84 mL, 10.4 mmol). Afterstirring at 40° C. for 12 h, the mixture was diluted with DCM and washedwith 1N HCl (2×10 mL), H₂O (10 mL), and brine (10 mL), then dried overNa₂SO₄ and concentrated. Purification via flash column chromatography onsilica gel gave 330 mg (36% yield over 2 steps) of the tosylate as acolorless oil.

A solution of the tosylate (203 mg, 0.453 mmol) in 3.1 mL of anhydrousacetone was treated with LiBr (385 mg, 4.53 mmol). After stirring at 60°C. for 8 h, the solvent was removed and the resulting residue wasdissolved in EtOAc. The organic mixture was washed with water, driedover MgSO₄, filtered, and concentrated. Purification via flash columnchromatography on silica gel gave 126 mg (78% yield) of the titlecompound as a colorless oil.

2-((3S,8S,9S,10R,13R,14S,17R)-10,13-Dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)ethanol(B-2-b)

A solution of cholesterol (1.50 g, 3.89 mmol) in 4 mL of DCM was treatedwith p-toluenesulfonyl chloride (1.49 g, 7.78 mmol), pyridine (4 mL),and DMAP (94.9 mg, 0.780 mmol). After stirring at RT for 12 h, themixture was diluted with DCM and washed with 1N HCl (2×5 mL), H₂O (5mL), and brine (5 mL), then dried over Na₂SO₄ and concentrated in vacuo.Recrystallization from chloroform and methanol gave 1.66 g (79% yield)of the tosylate intermediate as a white solid.

A microwave vial charged with the tosylate intermediate (500 mg, 0.926mmol) in 7.7 mL of 1,4-dioxane was treated with 2.6 mL of ethyleneglycol. After heating to 160° C. by microwave irradiation for 10 min,the solvent was removed, and the residue was dissolved in chloroform andwashed with saturated NaHCO₃ (5 mL), H₂O (5 mL), and brine (5 mL), thendried over Na₂SO₄ and concentrated in vacuo. Purification of the crudematerial by flash column chromatography using silica gel provided 270 mg(68% yield) of the title compound as a white solid.

2-Bromo-N-(14-((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxy)-3,6,9,12-tetraoxatetradecyl)acetamide(B-2-c)

A solution of B-2-b (219 mg, 0.509 mmol) in 2.5 mL of THF was treatedwith sodium hydride (60% dispersion in mineral oil, 20.4 mg, 0.509 mmol)and stirred for 30 min. The mixture was cooled to 0° C. and treated withB-2-a (139 mg, 0.392 mmol) in 2.5 mL of THF. After heating to 40° C. for6 h, the reaction was quenched with saturated NH₄Cl (aq) and extractedwith EtOAc. Purification of the crude material by flash columnchromatography using silica gel provided 101 mg (36% yield) of the etherintermediate as a colorless oil.

A solution of the ether intermediate (74 mg, 0.105 mmol) in 2.1 mL ofDCM was treated with 0.53 mL of TFA. After stirring at RT for 40 min,the mixture was concentrated in vacuo and dissolved in 10 mL DCM. A 4-mLaliquot of this solution was taken into a separate vial, concentrated toa 1-mL volume, cooled to 0° C., and charged with bromoacetic anhydride(13.6 mg, 0.053 mmol) and DIEA (15 μL, 0.088 mmol). After stirring at RTfor 12 h, the reaction mixture was directly purified by flash columnchromatography using silica gel to give 12.6 mg (41% over 2 steps) ofthe title compound as an colorless oil.

Example 3. Synthesis of Oxyntomodulin-Lipid Conjugate with Staple(Scheme C)

A solution of 1,4-butanediamine (0.18 g, 2 mmol) in DCM (10 mL) wastreated with bromoacetic anhydride (1.1 g, 4 mmol), DIEA (0.7 mL, 4mmol) at −20° C. The reaction mixture was then stirred for 2 h, and thesolvent was removed. Purification by flash column chromatography onsilica gel provided 0.53 g of L1 as a white solid (Yield 81%).

Peptide Cross-Linking with C4-Staple to Obtain Intermediate C-2.

The cross-linking reaction was carried out by incubating the purifieddicysteine-containing peptide C-1 with a mixed solvent solution 1.5equiv of 1,4-dibromoacetamide butane (L1) (1.5 equiv in 1:4 CH₃CN/30 mMNH₄HCO₃ buffer, pH 8.5) to obtain a final peptide concentration of 1 mM.The mixture was stirred at RT for 2 h. Under ice cooling, acetic acidwas then added dropwise to pH 5. Crude cross-linked peptide was thenpurified by preparative HPLC. The main peak was collected andlyophilized to afford C-2 as a powder with greater than 95% purity andin greater than 70% product yield. The molecular weight of peptide wasanalyzed by ESI-MS: calcd MW 4738.59; found 1185.6 [M/4+1]⁺, 948.7[M/5+1]⁺, 789.8 [M/6+1]⁺.

Deprotection of Thiol to Obtain Intermediate C-3.

The cross-linked peptide C-2 was treated with AgOTf (50 equiv) inanisole/TFA (1:50) to obtain a final peptide concentration of 1 mM. Themixture was stirred at 0° C. for 2 h. Ether (50-fold) was added and theresulting powder was collected by centrifugation. The powder wassuspended in 50% AcOH to obtain a final peptide concentration of 0.2 mM.DTT (100 equiv) was added. The mixture was stirred for 2 h in theabsence of light. After centrifugation, the supernatant was purified bypreparative HPLC. The fraction corresponding to the main peak wascollected and lyophilized to afford C-3 as a powder with greater than95% purity in about 35% product yield. The molecular weight of peptidewas analyzed by ESI-MS: calcd MW 4667.5; found 1167.7 [M/4+1]⁺, 934.6[M/5+1]⁺.

Conjugation with Lipid Derivative to Obtain Peptide Lipid Conjugate(C-4-a, C-4-b, C-4-c).

The Cys peptide intermediate C-3 was dissolved in DMSO and reacted withthe appropriate lipid derivative (with haloacetamide group) dissolved inTHF to obtain a final peptide concentration of 1 mM. The mixture wasstirred at RT for 1 h. The reaction was quenched by the addition of TFAto a final pH of 4, and directly purified by preparative HPLC. Thefraction corresponding to the main peak was collected and lyophilized togive the desired compound as a powder with greater than 95% purity inabout 65% product yield. The molecular weight of peptide was analyzed byESI-MS.

Example 4. Synthesis of Oxyntomodulin-Lipid Conjugate withLipid-Ornithine Staple Precursor (Scheme D)

tert-butyl (2-(2-(2-tetradecanamidoethoxy)ethoxy)ethyl)carbamate (D-1).

Myristic acid (0.46 g, 2 mmol) was dissolved in 5 mL of DMF. HATU (0.8g, 2.1 mmol) and DIEPA (0.4 mL, 2.2 mmol) was added followed by theaddition of tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (0.5g, 2 mmol). The reaction mixture was then stirred for 6 h, and thesolvent was removed. The product was extracted with EtOAc (3×15 mL). Theorganic layer was successively washed with sat. NaHCO₃, cooled HCl (1 M)and brine, dried over Na₂SO₄, filtered, and concentrated. Purificationby flash column chromatography on silica gel provided 0.81 g of desiredcompound as a white solid in 90% product yield. ESI-MS: calcd MW 458.4;found 459.6 [M+1]⁺.

di-tert-butyl(5,16-dioxo-9,12-dioxa-6,15-diazanonacosane-1,4-diyl)dicarbamate (D-2)

A solution of D-1 (0.46 g, 1 mmol) in DCM (10 mL) was treated with TFA(2 mL) for 2 h. The mixture was concentrated and followed by theaddition of Boc-Orn(Boc)-OH (0.33 g, 1 mmol), HATU (0.41 g, 1.1 mmol),DIEPA (0.4 mL, 2.2 mmol) in 5 mL of DMF. The reaction mixture was thenstirred for 6 h, and the solvent was removed. The product was extractedwith EtOAc (3×15 mL). The organic layer was successively washed withsat. NaHCO₃, cooled HCl (1 M) and brine, dried over Na₂SO₄, filtered,and concentrated. Purification by flash column chromatography on silicagel provided 0.58 g of desired compound as a white solid in 87% productyield. ESI-MS: calcd MW 672.9; found 673.6 [M+1]⁺.

(S)—N,N′-(5,16-dioxo-9,12-dioxa-6,15-diazanonacosane-1,4-diyl)bis(2-bromoacetamide)(D-3, Staple L2)

A solution of D-2 (0.33 g, 0.5 mmol) in DCM (10 mL) was treated with TFA(2 mL) for 2 h. The mixture was concentrated and followed by theaddition of bromoacetic anhydride (0.52 g, 1 mmol), DIEPA (0.4 mL, 2.2mmol) in 10 mL of DCM. The reaction mixture was then stirred for 6 h,and the solvent was removed. The product was extracted with EtOAc (3×15mL). The organic layer was successively washed with sat. NaHCO₃, cooledHCl (1 M) and brine, dried over Na₂SO₄, filtered, and concentrated.Purification by flash column chromatography on silica gel provided 0.59g of desired compound as a white solid in 83% product yield. ESI-MS:calcd MW 714.6; found 715.4 [M+1]⁺.

Peptide lipid conjugate D-4 was prepared in an analogous manner as C-2,substituting 1,4-dibromoacetamide butane with D-3. Purification bypreparative HPLC lyophilized to give the desired compound as a whitepowder with greater than 95% purity in about 55% product yield. Themolecular weight of peptide was analyzed by ESI-MS: calcd MW 4908.8;found 1228.0 [M/4+1]⁺, 982.6 [M/5+1]⁺, 819.0 [M/6+1]⁺.

Example 5. Preparation of Staple L3

tert-butyl(9,20-dioxo-3,6,13,16-tetraoxa-10,19-diazatritriacontyl)carbamate (E-1)

A solution of tert-butyl(2-(2-(2-tetradecanamidoethoxy)ethoxy)ethyl)carbamate (D-1) (0.23 g, 0.5mmol) in DCM (5 mL) was treated with TFA (1 mL) for 2 h. The mixture wasconcentrated and followed by the addition of2,2-dimethyl-4-oxo-3,8,11-trioxa-5-azatetradecan-14-oic acid (0.214 g,0.5 mmol), HATU (0.21 g, 0.55 mmol), DIEPA (0.2 mL, 1.1 mmol) in 2 m ofDMF. The reaction mixture was then stirred for 6 h, and the solvent wasremoved. The product was extracted with EtOAc (3×10 mL). The organiclayer was successively washed with sat. NaHCO₃, cooled HCl (1 M) andbrine, dried over Na₂SO₄, filtered, and concentrated. Purification byflash column chromatography on silica gel provided 0.25 g of desiredcompound as a white solid in 81% product yield.

(S)-di-tert-butyl(5,15,26-trioxo-9,12,19,22-tetraoxa-6,16,25-triazanonatriacontane-1,4-diyl)dicarbamate(E-2)

A solution of tert-butyl(9,20-dioxo-3,6,13,16-tetraoxa-10,19-diazatritriacontyl)carbamate (E-1)(0.15 g, 0.25 mmol) in DCM (5 mL) was treated with TFA (1 mL) for 2 h.The mixture was concentrated and followed by the addition ofBoc-Orn(Boc)-OH (85 mg, 0.25 mmol), HATU (105 mg, 0.275 mmol), DIEPA(0.1 mL, 0.5 mmol) in 2 mL of DMF. The reaction mixture was then stirredfor 6 h, and the solvent was removed. The product was extracted withEtOAc (3×10 mL). The organic layer was successively washed with sat.NaHCO₃, cooled HCl (1 M) and brine, dried over Na₂SO₄, filtered, andconcentrated. Purification by flash column chromatography on silica gelprovided 0.16 g of desired compound as a white solid in 77% productyield.

(S)—N,N′-(5,15,26-trioxo-9,12,19,22-tetraoxa-6,16,25-triazanonatriacontane-1,4-diyl)bis(2-bromoacetamide)(L3)

A solution of (S)-di-tert-butyl(5,15,26-trioxo-9,12,19,22-tetraoxa-6,16,25-triazanonatriacontane-1,4-diyl)dicarbamate(E-2) (125 mg, 0.15 mmol) in DCM (5 mL) was treated with TFA (2 mL) for2 h. The mixture was concentrated and followed by the addition ofbromoacetic anhydride (80 mg, 0.3 mmol), DIEPA (0.1 mL, 0.6 mmol) in 5mL of DCM at 0° C. The reaction mixture was then stirred for 6 h, andthe solvent was removed. The product was dissolved in 10 mL ofACN/H₂O/TFA (1:1:0.1) and purified by preparative HPLC (C18 column).Lyophilization provided the desired compound as a powder in 55% yieldover two steps.

Example 6. Preparation of Staple L4

(S)-di-tert-butyl (1-azido-13-oxo-3,6,9-trioxa-12-azaheptadecane-14,17-diyl)dicarbamate (F-1)

Boc-Orn(Boc)-OH (1.32 g, 4 mmol) was dissolved in 10 mL of DMF. HATU(1.7 g, 4.4 mmol) and DIEPA (0.75 mL, 4.4 mmol) were added followed bythe addition of 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanamine (0.88 g,4 mmol). The reaction mixture was then stirred for 16 h, and the solventwas removed. The product was purification by flash column chromatographyon silica gel provided 1.5 g of desired compound as a white solid in 75%product yield.

(S)-di-tert-butyl(1-amino-13-oxo-3,6,9-trioxa-12-azaheptadecane-14,17-diyl)dicarbamate(F-2)

A solution of (S)-di-tert-butyl(1-azido-13-oxo-3,6,9-trioxa-12-azaheptadecane-14,17-diyl)dicarbamate(F-1) (1.06 g, 2 mmol) in MeOH (20 mL) was treated with Pd/C (0.11 g)and 1 atm of H₂ for 6 h. The mixture was filtered and concentrated. Thecrude material was used immediately in the next step without furtherpurification.

(S)-tert-butyl9-((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,10,24-trioxo-3,14,17,20-tetraoxa-5,11,23-triazahentetracontan-41-oate(F-3)

Octadecanedioic acid mono-tert-butyl ester (0.22 g, 0.6 mmol) wasdissolved in 5 mL of DMF. HATU (0.23 g, 0.6 mmol) and DIEPA (0.1 mL, 0.6mmol) was added followed by the addition of (S)-di-tert-butyl(1-amino-13-oxo-3,6,9-trioxa-12-azaheptadecane-14,17-diyl)dicarbamate(F-2) (0.31 g, 0.6 mmol) in 5 mL of DMF. The reaction mixture was thenstirred for 24 h, and the solvent was removed. The product waspurification by flash column chromatography on silica gel provided 0.37g of desired compound as a white solid in 71% product yield.

(S)-1-bromo-7-(2-bromoacetamido)-2,8,22-trioxo-12,15,18-trioxa-3,9,21-triazanonatriacontan-39-oicAcid (L4)

A solution of (S)-tert-butyl9-((tert-butoxycarbonyl)amino)-2,2-dimethyl-4,10,24-trioxo-3,14,17,20-tetraoxa-5,11,23-triazahentetracontan-41-oate(F-3) (0.26 g, 0.3 mmol) in DCM (10 mL) was treated with TFA (5 mL) for6 h. The mixture was concentrated and followed by the addition ofbromoacetic anhydride (0.16 g, 0.6 mmol), DIEPA (0.2 mL, 1.2 mmol) in 10mL of DCM. The reaction mixture was then stirred for 2 h at 0° C., andthe solvent was removed. The product was dissolved in 10 mL ofACN/H₂O/TFA (1:1:0.1) and purified by preparative HPLC (C18 column).Lyophilization provided the desired compound as a powder in 45% yieldover two steps.

Example 7. Preparation of Staple L5

Preparation of Fmoc-Lys(ivDde)-2-chlorotrityl resin (G-1)

2-Chlorotrityl chloride resin (Novabiochem, 100-200 mesh, 1% DVB, loaded1.0 mmol/g) (5.0 g, 5 mmol) was swollen in DCM (80 mL) for 1 h and thendrained. Fmoc-Lys(ivDde)-OH (Novabiochem, 1 equiv) was suspended in DMF(30 mL) and DCM (30 mL) and DIPEA (1.1 equiv). This suspension was addedto the resin and shaken for 2 h. The resin was drained and washed withDMF (3×), DCM (3×) and treated with CH₃OH/DCM/DIPEA (8:1:1) for 30 minto cap the unreacted trityl chloride sites, washed with DCM (3×), anddried under vacuum, and stored in a desiccator until use.

C18-diacid-Lys(ivDde)-2-chlorotrityl Resin (G-2)

To G-1 (5 mmol) was added piperidine in DMF (20%, 100 mL). The mixturewas shaken for 30 min and drained. Another 50 mL of 20% piperidine wasadded and the mixture was shaken for another 30 min. Positive ninhydrintest was observed. The resin was then washed as described above for G-1.The resin was then treated with octadecanedioic acid mono-tert-butylester (AstaTech) (3 equiv, 15 mmol), HATU (3.3 equiv), and DIPEA (3.3equiv) in DMF (100 mL) for 2 h and repeated until a negative ninhydrintest was observed. The resin was washed with DMF and DCM as describedfor G-1 and used directly in the next step.

Staple L5

G-2 was treated twice with 2% hydrazine in DMF (100 mL, 15 min).Positive ninhydrin test was observed. The resin was then washed asdescribed for G-1 and treated with Fmoc-PEG2-propionic acid (QuantaBioDesign) (3 equiv) using HATU (3.3 equiv), and DIPEA (3.3 equiv) inDMF (100 mL) for 2 h. The resin was then washed as before. Then Fmocgroup was removed as described for G-2 and the above washing steps wererepeated. The resin was then treated with Fmoc-Orn(Fmoc)-OH(Novabiochem, 3 equiv), HATU (3.3 equiv), and DIPEA (3.3 equiv) in DMF(100 mL) for 2 h. The resin was again washed as described above. ThenFmoc group was removed and the above washing steps were repeated. Theresin was then treated with bromoacetic anhydride (4 equiv), and DIPEA(4.4 equiv) in 200 mL of DCM for 30 min. After washing with DCM (3×),the product was cleaved from the resin using 5 mL of 10% TFA in DCMcontaining 10% H₂O and 10% triisopropylsilane for 1 h. After cleavage,TFA was removed under reduced pressure. The resulting yellow residue waswashed several times with cold diethyl ether and was finally dried to acrude product as yellow powder. The crude peptide was dissolved in DMSO(5 mL) and this solution was diluted to a final volume of 100 mL with50% CH₃CN-water. The solution was filtered. The filtered solution (10mL) was loaded onto the preparative HPLC column (Phenomenex, Prep C18,300A, 50×250 mm) equilibrated with 10% CH₃CN (0.05% TFA) in water (0.05%TFA), and the column was eluted with 10% CH₃CN (0.05% TFA) in water(0.05% TFA) to wash DMSO from the column. The composition of the eluentthen was ramped to 35% CH₃CN-water (0.05% TFA) over 1 min, and a lineargradient was initiated at a rate of 1%/min of CH₃CN (0.05% TFA) intowater (0.05% TFA) and run for 50 min. Eluted fractions were checked forpurity on an analytical scale reversed phase C18 column (Phenomenex,C18, 120A, 4.6×50 mm) and fractions containing the product in >95%purity were combined and lyophilized to afford the title compound in 31%product yield. The molecular weight of product was analyzed by ESI-MS:calcd MW 1117.0; found 1118.3 [M+1]⁺, 1119.2 [M+2]⁺.

Example 8. Preparation of Staple L6

L6 was prepared in an analogous manner as L5 by substituting2-chlorotrityl chloride resin with Rink Amide MBHA resin (Novabiochem).Purification by preparative HPLC and lyophilization gave the titlecompound as a white powder with greater than 95% purity in about 35%product yield. The molecular weight of peptide was analyzed by ESI-MS:calcd MW 1116.0; found 1117.2 [M+1]⁺, 1118.7 [M+2]⁺.

Example 9. Preparation of Staple L23

L22 was prepared in an analogous manner as L5 by starting with2-chlorotrityl chloride resin and mono-Fmoc ethylene diaminehydrochloride (Novabiochem). Purification by preparative HPLC andlyophilization gave the title compound as a white powder with greaterthan 95% purity in about 15% product yield. The molecular weight ofpeptide was analyzed by ESI-MS: calcd MW 1159.1; found 1160.2 [M+1]⁺.

Example 10. General Procedure of the Cross-Linking Reaction Using aStaple Via Thioether Linkage

The cysteine side chain thiols were then derivatized with haloacetamides(L1 to L22) to form the bis-thioether peptidomimetic macrocycles. Thecross-linking reaction was carried out by incubating the peptide with amixed solvent solution 1.5 equiv of the linker in 1:4 CH₃CN/30 mMNH₄HCO₃ buffer, pH 8.5) to obtain a final peptide concentration of 1 mM.The mixture was stirred at rt for 2 h. Under ice cooling, acetic acidwas then added dropwise to pH 5. Crude cross-linked peptide was thenpurified by preparative HPLC (C18 column). Lyophilization gave thedesired peptide as a white solid.

FIG. 7 depicts two macrocyclization routes to obtain the desired mTAs.The staple, with or without one or more attached HEMS, is depicted as L.

Additional compounds in Tables 1, 2, and 6-9 may be prepared usinganalogous procedures.

TABLE 1 entry Structure*  1

 2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

*X can be Cl, I, Br, maleimide, an amino acid which is part of thepeptide, or a staple; Y can be OH, an amino acid which is part of thepeptide, or a staple; Z can be S which is part of an amino acid which ispart of the peptide, S which is part of the staple, or S which is partof a lipid derivative (to form a disulfide).

TABLE 2 en- Sta- try ple Structure 35 L1 

36 L2 

37 L3 

38 L4 

39 L5 

40 L6 

41 L7 

42 L8 

43 L9 

44 L10

45 L11

46 L12

47 L13

48 L14

49 L15

50 L16

51 L17

52 L18

53 L19

54 L20

55 L21

56 L22

Example 11. Generation of CREB Responsive Luciferase Stable Cell LinesOverexpressing Glucagon, GLP-1, GIP, or GLP2 Receptor

CREB responsive luciferase stable HEK 293 cell line overexpressing humanglucagon receptor (GCGR), glucagon-like peptide 1 receptor (GLP-1R),Glucose-dependent insulinotropic polypeptide receptor (GIPR), orGlucagon-like peptide 2 receptor (GLP-2R) was generated as follows.

HEK293 cells were infected with lent virus encoding firefly luciferasegene under the control of CRE promoter, as described in the manual(Qiagen, Netherlands) and then were selected using 1 μg/mL puromycin(Life technologies, Carlsbad) for 1 week. The survived cells were namedas CRE-HEK293, expanded and then transfected with a G418 selectivemammalian expression plasmid encoding human GCGR, GLP-1R, GIPR orGLP-2R. In brief, GCGR, GLP-1R, GIPR, or GLP-2R plasmid was transfectedinto CRE-HEK293 cells using Lipofectamine 2000 and selected with 400μg/mL geneticin (Life technologies, Carlsbad, Calif.). Single colonystable cell lines overexpressing both CRE-luciferase and GCGR, GLP-1R,GIPR, or GLP-2R were then established for in vitro activity assays.These four stable cell lines were named as HEK293-GCGR-CRE,HEK293-GLP-1R-CRE, HEK293-GIPR-CRE, and HEK293-GLP-2R-CRE.

Example 12. In Vitro Activity Assays (Receptor-Mediated cAMP Synthesis)

HEK293-GCGR-CRE, HEK293-GLP-1R-CRE, HEK293-GIPR-CRE, andHEK293-GLP-2R-CRE cells were seeded in a white 384-well plate at adensity of 5,000 cells per well and cultured for 24 hours. Cells weretreated with different peptides in a dose dependent manner. On the nextday, 10 μl of Bright-Glo reagent (Promega, Madison, Wis.) were addedinto each well and firefly luminescence was determined using an Envisionmultilabel plate reader (PerkinElmer, Waltham, Mass.). EC₅₀ of eachpeptide was calculated using GraphPad Prism 6 software (GraphPad, SanDiego, Calif.).

Exemplary data for select mTAs from Tables 6-9 are shown in FIGS. 8-11.

Example 13. In Vivo Pharmacokinetic (PK) Studies

Female CD-1 mice obtained from Charles River Limited were used afterovernight food deprivation for in vivo PK study. Peptides were dissolvedin pH adjusted Phosphate buffered saline (PBS). 100 μL of each peptide(0.3 mg/kg) was administrated into each mouse through either i.v. or s.croute. Food was provided to mice immediately after bleeding at 30minute. Blood were extracted into heparinized tubes and centrifuged at3,000×g for 15 min. The resulting supernatant plasma were then stored at−80° C. for activity assays. The concentrations of peptides in plasma ateach time point were determined by activity assay described above and invivo half-life of each peptide was calculated by using Winnonlin Phoenixsoftware (Pharsight Corp, St. Louis, Mo.).

Exemplary data are for select mTAs from Tables 6-9 are shown in FIG. 12.The curves represent the mean of five animals. Calculated half-lifevalues are shown.

Example 14. Oral Glucose Tolerance Test (OGTT) and IntraperitonealGlucose Tolerance Test (IPGTT)

CD1 mice were fasted for overnight and then administrated with certainamount of peptides through either i.v. or s.c. route. After 6 hours,mice were orally or intraperitoneally administrated with bolus dose ofglucose solution at 2 g/kg body mass at concentration of 100 mg/mL andtheir tail blood glucose levels were measured before (0 min) and afterglucose challenge for 2 to 3 hours.

Exemplary data for mTA4 and mTA37 (see Table 6) are shown in FIG. 13(n=5 for both). Both mTA4 and mTA37 are functional in wild-type mice.The data indicate increased oral glucose tolerance after administrationof mTA. OGTT results for mTA4 demonstrated that mTA4 had a greatereffect than exendin-4 at 6 hours after a single i.v. dose of mTA.

Example 15. Body Weight, Food Intake, and Visceral Fat Mass Measurement

Diet Induced Obese (DIO) mice were purchased from Charles River andadministrated by s.c. route with GLP1R agonist or GLP1R/GCGR dualagonist. Mouse body weight and food intake were monitored daily for 2weeks, and followed before (5 days in total) and during treatment(5-weeks in total). After 5 weeks, mice were sacrificed and visceral fatmass were taken out and weighed.

Dose dependent weight loss induced by mTA4 or mTA37 (see Table 6) isshown in FIG. 14. Chronic effect on body weight loss with dailyadministration of mTA4 or mTA37 for 5 weeks and cumulative food intakefor 2 weeks are shown in FIG. 15. Increased intra-peritoneal glucosetolerance after daily administration of mTA4 or mTA37 for 5 weeks isshown in FIG. 16. Reduced fasting blood glucose levels after dailyadministration of mTA4 or mTA37 for 5 weeks is shown in FIG. 17.

Example 16. Cholesterol Level Determination

Collected plasma was used for cholesterol level determination accordingto the manufacturer's guide (cholesterol assay kit, Abcam, Cambridge,England). Briefly, plasma was diluted using cholesterol assay buffer andthen reacted with same volume of reaction mix containing cholesterolassay buffer, cholesterol probe, enzyme mix and cholesterol esterase.After incubation at 37° C. for 1 hour, the absorbance at 560 nm wasmeasured using an Envision multilabel plate reader (PerkinElmer,Waltham, Mass.). Subsequently, the concentration of cholesterol inplasma was calculated using a standard curve.

Exemplary data for select mTAs is shown in FIG. 18B. There was decreasedcholesterol level in plasma after daily administration of mTA4 or mTA37for 5 weeks.

Example 17. Triglyceride Level Measurement

Collected plasma was again used for triglyceride level determinationusing a triglyceride colorimetric assay kit (Cayman chemical, Ann Arbor,Mich.). 5 μL of plasma samples or standard were added into a 384 wellplate and followed by adding 75 μL of diluted enzyme buffer to eachwell. The plate was incubated at room temperature for 15 min, and theabsorbance was read at 560 nm using an Envision multilabel plate reader(PerkinElmer, Waltham, Mass.). Again, the concentration of triglyceridein plasma was calculated using a standard curve.

Exemplary data for select mTAs is shown in FIG. 18A. There was decreasedtriglyceride level in plasma after daily administration of mTA4 or mTA37for 5 weeks.

Example 18. Oil Red O Staining (Lipid Droplet Staining)

Frozen tissue sections of liver were cut at 10 m and air dried to theslides. After fixation in 10% formalin for 5 min, the slides werebriefly washed with running tap water for 10 min, followed by rinse with60^ isopropanol. Subsequently, oil red O working solution (0.3% oil redO) was used for lipid staining for 15 min. Slides were again rinsed with60% isopropanol and then nuclei were lightly stained with alumhaematoxylin, followed by rinse with distilled water and mounted inglycerine jelly. After half an hour, pictures were taken undermicroscopy.

Exemplary data are shown in FIG. 19, in which reduced lipid dropletamounts were observed after daily administration of mTA4 or mTA37 for 5weeks.

Example 19. Application of Micro-Needle Based Patch on Guinea Pigs

Microneedle patch based transdermal drug delivery was performed usingguinea pigs. Guinea pigs were shaved one day before patch application.On the second day, micro-needle based patches were applied onto theanimal skin for 5 min and blood was extracted at different time points(5 min, 30 min, 1 h, 2 h, 3 h, 5 h, 8 h, 24 h, 32 h, 48 h, 72 h).Peptide concentration at each time point was determined using the samemethod (functional activity assay) as described above. Transdermalbioavailability (F) was calculated as the ratio of area under the curve(AUC) between microneedle patch application and i.v. injection groups.

Exemplary data are shown in FIGS. 20-22. Pharmacokinetic profiles ofmTA4 after i.v. or s.c. injection or micro-needle based transdermaldelivery are shown in FIG. 20. Blood glucose curves and AUC values froman oral glucose tolerance test at 24 h, 48 h, and 96 h after mTAadministration through micro-needle based transdermal delivery are shownin FIGS. 21 and 22.

Example 20. Murine Model of Ulcerative Colitis (DSS Model)

Colitis in mouse was induced by adding 3% DSS (dextran sulfate sodium)in the drinking water for 12 consecutive days. Besides DSS placement,mice were daily treated with GLP-2 analogs (40 μg/kg/day) [GLP-2G is theGLP2 sequence with G2S mutation and is a known drug called teduglutide].Cyclosporine A (20 mg/kg/day) was used for the positive control groupand PBS for the negative control group. During the experiment period,body weight was measured every day.

Intestinal Weight/Body Weight Measurement

Animals were sacrificed after 12 days of treatment. Small intestine wasimmediately excited and flushed with PBS. After PBS was gently squishedout, intestinal weight was weighed using an analytical balance.Intestinal vs. body weight ratio was then calculated and analyzed.

Body weight and intestine weight versus body weight in DSS-inducedcolitis mice after daily administration of mTA68 for 12 days are shownin FIG. 23.

Example 21. Efficacy and Safety of mTA for the Treatment of Diabetes andObesity

Purpose: Different doses of mTA are compared to placebo to determineefficacy and safety for the treatment of patients with diabetes andobesity.

Condition Intervention Phase Diabetes and Obesity Drug: single agonistor dual agonist Phase 1 mTA Drug: Placebo Phase 1

Study Type: Interventional

Study Design: Allocation: Randomized

Endpoint Classification: Safety/Efficacy Study

Intervention Model: Parallel Assignment

Masking: Double Blind (Subject, Caregiver, Investigator, OutcomesAssessor)

Primary Purpose: Treatment

Primary Outcome Measures: reduced blood glucose, reduced body weight andHbA1C [Time Frame: Up to 52 weeks] [Designated as safety issue: No]

Secondary Outcome Measures: improved metabolic profiles

[Time Frame: Up to day 52 weeks] [Designated as safety issue: No]

Arms Assigned Intervention Placebo Comparator: Placebo Drug: Placebosubcutaneous or microneedle patch subcutaneous or microneedle deliverypatch delivery Experimental: single and dual Drug: single and dualagonist mTA agonist mTA through subcutaneous through subcutaneous ormicroneedle or microneedle patch delivery patch delivery

This is an international, randomized, double-blind, placebo-controlled,Phase II/III trial of single and dual agonist mTA for the treatment ofdiabetes and obesity.

TABLE 3 Therapeutic Agents (TAs)-Amino acid sequences NAME SEQ ID NOSEQUENCE Oxyntomodulin 1 HSQGTFTSDYSKYLDSRRAQDFVQ WLMNTKRNRNNIAExendin-4 or 2 HGEGTFTSDLSKQMEEEAVRLFIE Exenatide WLKNGGPSSGAPPPS hGLP-13 HAEGTFTSDVSSYLEGQAAKEFIA WLVKGR Glucagon 4 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT hGLP-2 5 HADGSFSDEMNTILDNLAARDFIN WLIQTKITD hGIP 6YAEGTFISDYSIAMDKIHQQDFVN WLLAQKGKKNDWKHNITQ

TABLE 4 Therapeutic Agents-Amino acid sequences NAME SEQ ID NO SEQUENCEOxm-1  7 HsQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIAC Oxm-i + 4  8HsQGTFTSDYSKYLDERAACEFICWLMNTKRNRNNIA Oxm-i + 11  9HsQGTFTSDYSKCLDERAVRLFICWLMNTKRNRNNIA OXM-i + 14 10HsQGTFTSDCSKYLDERAVRLFICWLMNTKRNRNNIA OXM-2t + 1t 11HsQGTFTSCYSKCLDECAVRLFICWLMNTKRNRNNIA OXM-i + 15 12HsQGTFTSCYSKCLDERAVRLFICWLMNTKRNRNNIA Ex-i + 7 13HGEGTFTSDLSKQMEECAVRLFICWLKNGGPSSGAPPPS Ex-i + 7-Cys 14HGEGTFTSDLSKQMEECAVRLFICWLKNGGPSSGAPPPSC Ex-i + 4 15HGEGTFTSDLSKQMEEEAVCLFICWLKNGGPSSGAPPPS Ex-i + 11 16HGEGTFTSDLSKCMEEEAVRLFICWLKNGGPSSGAPPPS Ex-i + 14 17HGEGTFTSDCSKQMEEEAVRLFICWLKNGGPSSGAPPPS Ex-i + 15 18HGEGTFTSCLSKQMEEEAVRLFICWLKNGGPSSGAPPPS Ex-2t + 1t 19HGEGTFTSCLSKCMEEEAVRLFICWLKNGGPSSGAPPPS Dual-2a 20HsQGTFTSDYSKYLDSKAAHDFVCWLLRA Dual-2b 21 HSQGTFTSDYSKYLDSCAAHDFVCWLLRADual-2c 22 HsQGTFTSDYSKCLDSKAAHDFVCWLLRA Dual-2d 23HsQGTFTSCYSKYLDSKAAHDFVCWLLRA Dual-2e 24 HsQGTFTSCYSKCLDSCAAHDFVCWLLRADual-3a 25 YXEGTFTSDYSIYLDKQAACEFVCWLLAGGPSSGAPPPSK Dual-3b 26YXEGTFTSDYSIYLDKCAAXEFVCWLLAGGPSSGAPPPSK Dual-3c 27YXEGTFTSDYSICLDKQAAXEFVCWLLAGGPSSGAPPPSK Dual-3d 28YXEGTFTSDCSIYLDKQAAXEFVCWLLAGGPSSGAPPPSK Dual-3e 29YXEGTFTSCYSICLDKQAAXEFVCWLLAGGPSSGAPPPSK Lowercase letters representD-amino acids

TABLE 5 Therapeutic Agents: Modified Therapeutic Peptides NAME SEQ ID NOSEQUENCE^(a) Exendin-4 (A) 30 HGEGTFTSDLSKQMEEXAVRLFIXWLKNGGPSSGAPPPSExendin-4 (B) 31 HGEGTFTSDLSKXMEEEAVRLFIXWLKNGGPSSGAPPPS Exendin-4 (C)32 HGEGTFTSDXSKQMEEEAVRLFIXWLKNGGPSSGAPPPS OXM (A) 33HsQGTFTSDYSKYLDSXRAQDFVXWLMNTKRNRNNIA OXM (B) 34HsQGTFTSDYSKYLDEXAAKEFIXWLMNTKRNRNNIA GLP-1/Glucagon (A) 35HsQGTFTSDYSKYLDSXAAHDFVXWLLRA GLP-1/Glucagon (B) 36HsQGTFTSDYSKYLDEXAAKEFIXWLLRA GLP-1/Glucagon (C) 37HsQGTFTSDYSKYLDEXAAKEFIXWLLRAGPSSGAPPPS GLP-1/Glucagon (D) 38HsQGTFTSDYSKYLDEXAAKEFIXWLLNGGPSSGAPPPS GLP-1/GIP (A) 39YaEGTFTSDYSIYLDKXAAKEFVXWLLAGGPSSGAPPPSK GLP-1/GIP/ 40YaEGTFISDYSKYLDEXAAKEFIXWLMNTKRNRNNIA Glucagon (A) GLP-1/GIP/ 41HAibQGTFTSDKSKYLDEXAAQDFVXWLLDGGPSSGAPPPS Glucagon (B) GLP-2 (A) 42HGDGSFSDEMNTILDNXAARDFIXWLIQTKITD GLP-2 (B) 43HGDGSFSDELLTILDLXAARDFIXWLIQTKITD GLP-2 (C) 44HGDGSFSDEMNTILDXLAARDFIXWLIQTKITD GLP-2 (D) 45HGDGSFSXEMNTILXALAARDFINWLIQTKITD GLP-2 (E) 46HGDGSFSDEMNTILDALAARXFINWLIXTKITD GLP-2 (F) 47HGDGSFSDXMNTILDXLAARDFINWLIQTKITD GLP-2 (G) 48HGDGSFSDEMXTILDNLXARDFINWLIQTKITD GLP-2 (H) 49HGDGSFSDEMNTILDNXAARDFIXWLIQTKITDPSSGAPPPS GLP-2 (I) 50HGDGSFSDXMNTILDXLAARDFINWLIQTKITDPSSGAPPPS GLP-2 (J) 51HGDGSFSDEMXTILDNLXARDFINWLIQTKITDPSSGAPPPS GLP-1 (A) 52HGEGTFTSDVSSYLEGXAAKEFIXWLVKGR GLP-1 (B) 53HAibEGTFTSDVSSYLEGXAAKEFIXWLVKGR ^(a) X is cysteine.

TABLE 6 Modified Therapeutic Agents Mass Mass GLP-1R GCGR mTAStructure^(a) Expected Found (EC₅₀, nM) (EC₅₀, nM) t_(1/2) (h) 1Exendin-4 (A) and L1 4302.9 1076.2 0.03 ND ND ([M + 4H]⁴⁺), 861.4 ([M +5H)⁵⁺) 2 Exendin-4 (A) and L2 4687.5 1172.8 0.03 ND ND ([M + 4H]⁴⁺),938.4 ([M + 5H]⁵⁺) 3 Exendin-4 (A) and L4 4817.6 1205.3 0.03 ND ND ([M +4H]⁴⁺), 964.4 ([M + 5H]⁵⁺) 4 Exendin-4 (A) and L5 5089.9 1273.2 0.03 NDt_(1/2) = 13 h ([M + 4H]⁴⁺), (i.v.), 1018.7 C_(max) = 4 h ([M + 5H]⁵⁺)(s.c.) 5 Exendin-4 (B) and 4331.5 1444.7 0.05 ND ND L14 ([M + 3H]³⁺),1083.8 ([M + 4H]⁴⁺) 6 Exendin-4 (B) and 4345.5 1449.5 0.03 ND ND L15([M + 3H]³⁺), 1087.4 ([M + 4H]⁴⁺) 7 Exendin-4 (B) and 4359.5 1454.1 0.03ND ND L16 ([M + 3H]³⁺), 1090.9 ([M + 4H]⁴⁺) 8 Exendin-4 (B) and 4373.51458.9 0.03 ND ND L17 ([M + 3H]³⁺), 1094.4 ([M + 4H]⁴⁺) 9 Exendin-4 (C)and 4375.1 1458.9 0.53 ND ND L16 ([M + 3H]³⁺), 1094.4 ([M + 4H]⁴⁺) 10Exendin-4 (C) and 4403.2 1101.8 2.5 ND ND L18 ([M + 4H]⁴⁺), 881.6 ([M +5H]⁵⁺) 11 Exendin-4 (C) and 4431.2 1108.8 0.15 ND ND L19 ([M + 4H]⁴⁺),887.2 ([M + 5H]⁵⁺) 12 Exendin-4 (C) and 4422.1 1106.2 0.62 ND ND L20([M + 4H]⁴⁺), 885.2 ([M + 5H]⁵⁺) 13 Exendin-4 (C) and 4450.2 1113.6 0.34ND ND L21 ([M + 4H]⁴⁺), 890.8 ([M + 5H]⁵⁺) 14 OXM (A) and L1 4539.21135.8 ND ND ND ([M + 4H]⁴⁺), 908.8 ([M + 5H]⁵⁺) 15 OXM (B) and L14524.3 1132.2 0.03 0.2 ND ([M + 4H]⁴⁺), 906.7 ([M + 5H]⁵⁺) 16 OXM (B)and L2 4908.8 1228.0 0.04 0.3 t_(1/2) = 2 h ([M + 4H]⁴⁺), (i.v.), 982.6C_(max) = 8 h ([M + 5H]⁵⁺) (s.c.) 17 OXM (B) and L4 5038.9 1260.7 2.0 3ND ([M + 4H]⁴⁺), 1008.8 ([M + 5H]⁵⁺) 18 OXM (B) and L5 5311.23 1328.80.20 NA ND ([M + 4H]⁴⁺), 1063.3 ([M + 5H]⁵⁺) 19 OXM (B) and L6 5310.21328.6 0.20 NA ND ([M + 4H]⁴⁺), 1063.1 ([M + 5H]⁵⁺) 20 OXM (B) and L74596.3 1152.6 17 5.4 ND ([M + 4H]⁴⁺), 922.4 ([M + 5H]⁵⁺) 21 OXM (B) andL8 4562.3 1141.6 0.12 0.7 ND ([M + 4H]⁴⁺), 913.4 ([M + 5H]⁵⁺) 22 OXM (B)and L9 4590.3 1148.6 40 14 ND ([M + 4H]⁴⁺), 919.2 ([M + 5H]⁵⁺) 23 OXM(B) and L10 4582.3 1146.6 ND ND ND ([M + 4H]⁴⁺), 917.4 ([M + 5H]⁵⁺) 24OXM (B) and L11 4496.2 1125.1 0.20 3 ND ([M + 4H]⁴⁺), 890.2 ([M + 5H]⁵⁺)25 OXM (B) and L12 4510.2 1128.6 0.10 0.5 ND ([M + 4H]⁴⁺), 902.2 ([M +5H]⁵⁺) 26 OXM (B) and L13 4538.3 1135.6 0.05 0.5 ND ([M + 4H]⁴⁺), 908.8([M + 5H]⁵⁺) 27 GLP-1/Glucagon (A) 3489.9 1164.0 0.2 0.04 ND and L1([M + 4H]⁴⁺), 873.3 ([M + 5H]⁵⁺) 28 GLP-1/Glucagon (A) 3874.5 1292.6 0.10.1 ND and L2 ([M + 4H]³⁺), 969.8 ([M + 5H]⁵⁺) 29 GLP-1/Glucagon (B)3551.0 1184.5 0.03 0.03 ND and L1 ([M + 3H]³⁺), 888.7 ([M + 4H]⁴⁺) 30GLP-1/Glucagon (B) 3935.6 1312.8 0.03 0.05 ND and L2 ([M + 3H]³⁺), 984.7([M + 4H]⁴⁺) 31 GLP-1/Glucagon (B) 4065.7 1356.8 0.03 0.12 ND and L4([M + 3H]³⁺), 1017.4 ([M + 4H]⁴⁺) 32 GLP-1/Glucagon (B) 4338.0 1446.60.3 1.0 ND and L5 ([M + 3H]³⁺), 1085.3 ([M + 4H]⁴⁺) 33 GLP-1/Glucagon(B) 4337.04 1446.7 0.05 0.4 t_(1/2) = 8 h and L6 ([M + 3H]³⁺), (i.v.),1085.4 C_(max) = 8 h ([M + 4H]⁴⁺) (s.c.) 34 GLP-1/Glucagon (C) 4900.81226.2 0.04 0.4 ND and L4 ([M + 4H]⁴⁺), 981.4 ([M + 5H]⁵⁺) 35GLP-1/Glucagon (C) 5172.9 1294.2 0.5 1 ND and L5 ([M + 4H]⁴⁺), 1035.6([M + 5H]⁵⁺) 36 GLP-1/Glucagon (C) 5171.9 1293.7 0.3 0.5 ND and L6 ([M +4H]⁴⁺), 1035.4 ([M + 5H]⁵⁺) 37 GLP-1/Glucagon (D) 4844.7 1212.2 0.03 0.2t_(1/2) = 8 h and L4 ([M + 4H]⁴⁺), (i.v.), 969.9 C_(max) = 4~8 h ([M +5H]⁵⁺) (s.c.) 38 GLP-1/Glucagon (D) 5117.0 1280.2 0.04 0.4 ND and L5([M + 4H]⁴⁺), 1024.4 ([M + 5H]⁵⁺) 39 GLP-1/Glucagon (D) 5115.2 1279.80.04 0.3 ND and L6 ([M + 4H]⁴⁺), 1024.1 ([M + 5H]⁵⁺) ^(a)Structure isdescribed by product of reaction between the peptide from Table 5 (X iscysteine) and the staple from Table 2.

TABLE 7 Modified Therapeutic Agents Mass Mass GLP-1R GIP-R mTAStructure^(a) Expected Found (EC₅₀, nM) (EC₅₀, nM) t_(1/2) (h) 40GLP-1/GIP (A) and 4396.2 1099.8 0.02 0.020 ND L1 ([M + 4H]⁴⁺), 879.7([M + 5H]⁵⁺) 41 GLP-1/GIP (A) and 4939.9 1646.8 0.02 0.06 t_(1/2) = 2.7h L3 ([M + 3H]³⁺), (i.v.), 1235.4 C_(max) = 2 h ([M + 4H]⁴⁺) (s.c.) 42GLP-1/GIP (A) and 4910.8 1228.7 ND ND ND L4 ([M + 4H]⁴⁺), 983.2 ([M +5H]⁵⁺) 43 GLP-1/GIP (A) and 5183.1 1727.9 0.5 1.1 ND L5 ([M + 3H]³⁺),1296.3 ([M + 4H]⁴⁺) ^(a)Structure is described by product of reactionbetween the peptide from Table 5 (X is cysteine) and the staple fromTable 2.

TABLE 8 Modified Therapeutic Agents Mass Mass GLP-1R GIP-R GCGR mTAStructure^(a) Expected Found (EC₅₀, nM) (EC₅₀, nM) (EC₅₀, nM) 44GLP-1/GIP/Glucagon 4547.3 1137.8 9.7 0.04 1.6 (A) and L1 ([M + 4H]⁴⁺),910.6 ([M + 5H]⁵⁺) 45 GLP-1/GIP/Glucagon 4271.6 1068.8 ND ND ND (B) andL1 ([M + 4H]⁴⁺), 855.3 ([M + 5H]⁵⁺) ^(a)Structure is described byproduct of reaction between the peptide from Table 5 (X is cysteine) andthe staple from Table 2.

TABLE 9 Modified Therapeutic Agents Mass GLP-2R mTA Structure^(a)Expected Mass Found (EG₅₀, nM) t_(1/2) (h) 46 GLP-2 (A) and L1 3898.51300.2 ([M + 3H]³⁺), 0.045 ND 975.3 ([M + 4H]⁴⁺) 47 GLP-2 (A) and L24282.9 1428.3 ([M + 3H]³⁺), 0.041 t_(1/2) = 2.3 h 1071.5 ([M + 4H]⁴⁺)(i.v.), C_(max) = 2 h (s.c.) 48 GLP-2 (A) and L3 4442.1 1481.9 ([M +3H]³⁺), 0.045 ND 1111.3 ([M + 4H]⁴⁺) 49 GLP-2 (A) and L4 4413.0 1103.3([M + 4H]⁴⁺), 0.241 ND 882.6 ([M + 5H]⁵⁺) 50 GLP-2 (A) and L5 4685.31172.3 ([M + 4H]⁴⁺), 6.4 ND 938.1 ([M + 5H]⁵⁺) 51 GLP-2 (A) and L64684.3 1171.2 ([M + 4H]⁴⁺), 0.7 ND 936.9 ([M + 5H]⁵⁺) 52 GLP-2 (C)and L13897.6 1300.2 ([M + 3H]³⁺), 5.6 ND 975.2 ([M + 4H]⁴⁺) 53 GLP-2 (D) andL1 3852.6 1284.4 ([M + 3H]³⁺), 14.3 ND 963.2 ([M + 4H]⁴⁺) 54 GLP-2 (E)and L1 3839.6 1280.9 ([M + 3H]³⁺), 0.13 ND 960.7 ([M + 4H]⁴⁺) 55 GLP-2(F) and L1 3882.6 1295.2 ([M + 3H]³⁺), 0.064 ND 971.5 ([M + 4H]⁴⁺) 56GLP-2 (F) and L5 4669.6 1168.4 ([M + 3H]³⁺), 0.77 ND 934.2 ([M + 4H]⁴⁺)57 GLP-2 (G) and L1 3940.6 1314.5 ([M + 3H]³⁺), 0.046 ND 986.2 ([M +4H]⁴⁺) 58 GLP-2 (G) and L5 4727.6 1182.1 ([M + 4H]⁴⁺), 7.98 ND 946.5([M + 5H]⁵⁺) 59 GLP-2 (H) and L1 4676.4 1169.9 ([M + 4H]⁴⁺), 0.064 ND936.3 ([M + 5H]⁵⁺) 60 GLP-2 (H) and L2 5060.9 1266.2 ([M + 4H]⁴⁺), 0.068ND 1012.3 ([M + 5H]⁵⁺) 61 GLP-2 (H) and L3 5220.1 1306.2 ([M + 4H]⁴⁺),0.068 ND 1045.1 ([M + 5H]⁵⁺) 62 GLP-2 (H) and L4 5191.1 1298.8 ([M +4H]⁴⁺), ND ND 1039.2 ([M + 5H]⁵⁺) 63 GLP-2 (H) and L5 5463.4 1366.7([M + 4H]⁴⁺), 2.66 ND 1093.3 ([M + 5H]⁵⁺) 64 GLP-2 (H) and L6 5462.21366.4 ([M + 4H]⁴⁺), 0.7 ND 1093.3 ([M + 5H]⁵⁺) 65 GLP-2 (I) and L35204.2 1302.2 ([M + 4H]⁴⁺), 0.06 t_(1/2) = 3.1 h 1040.6 ([M + 5H]⁵⁺)(i.v.), C_(max) = 3 h (s.c.) 66 GLP-2 (I) and L4 5175.1 1294.8 ([M +4H]⁴⁺), ND ND 1035.1 ([M + 5H]⁵⁺) 67 GLP-2 (I) and L5 5447.4 1362.9([M + 4H]⁴⁺), 3.1 ND 1090.6 ([M + 5H]⁵⁺) 68 GLP-2 (J) and L3 5262.21316.6 ([M + 4H]⁴⁺), 0.028 t_(1/2) = 4.7 h 1053.4 ([M + 5H]⁵⁺) (i.v.),C_(max) = 3 h (s.c.) 69 GLP-2 (J) and L4 5233.2 1309.3 ([M + 4H]⁴⁺), NDND 1047.6 ([M + 5H]⁵⁺) 70 GLP-2 (J) and L5 5505.5 1377.7 ([M + 4H]⁴⁺),8.8 ND 1102.2 ([M + 5H]⁵⁺) ^(a)Structure is described by product ofreaction between the peptide from Table 5 (X is cysteine) and the staplefrom Table 2.

What is claimed is:
 1. A modified therapeutic agent (mTA) comprising atherapeutic agent comprising the amino acid sequence selected from thegroup consisting of SEQ ID NOs: 30-32 and 35-53, a staple, and ahalf-life extending molecule, wherein the therapeutic agent iscovalently attached to the staple via two amino acid residues in thetherapeutic agent; the half-life extending molecule is covalentlyattached to the staple; and the half-life of the mTA is longer than thehalf-life of the therapeutic agent alone.
 2. The mTA of claim 1, whereinthe half-life extending molecule comprises a lipid, a polyglycol regionselected from

wherein m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20; or a combination thereof.
 3. The mTAof claim 2, wherein the lipid is selected from a group consisting ofsterols, bile acids, vitamin E, fatty di-acids, fatty acids, fattyamides, fatty amines, fatty alcohols, and derivatives thereof.
 4. ThemTA of claim 1, wherein at least one of the two amino acid residues isan amino acid addition or substitution on the therapeutic agent.
 5. ThemTA of claim 1, wherein each of the two amino acid residues is cysteine.6. The mTA of claim 1, wherein the staple comprises a group selectedfrom:

wherein each X is independently selected from O, NH, and S; each Y isindependently selected from N and CH; each Z is independently selectedfrom N and CH; each R⁸ and R⁹ is independently H, —C(O)(alkyl), orcomprises the half-life extending molecule; each R¹⁰ and R¹¹ isindependently oxo or comprises the half-life extending molecule; R¹² isindependently hydroxy, alkoxy, or comprises the half-life extendingmolecule; a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or17; b is 1, 2, 3, 4, 5, or 6; and c is 1 or
 2. 7. The mTA of claim 6,wherein the half-life extending molecule comprises a lipid, a polyglycolregion selected from

wherein m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20; or a combination thereof.
 8. The mTAof claim 7, wherein the half-life extending molecule comprises a lipidand a polyglycol region selected from

wherein m and n are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or
 20. 9. The mTA of claim 8, wherein thelipid is selected from a group consisting of sterols, bile acids,vitamin E, fatty di-acids, fatty acids, fatty amides, fatty amines,fatty alcohols, and derivatives thereof.
 10. The mTA of claim 1, whereinthe half-life of the mTA is 5-fold longer than the half-life of thetherapeutic agent alone.
 11. A pharmaceutical composition comprising themTA of claim 1 and a pharmaceutically acceptable excipient.
 12. The mTAof claim 6, wherein the staple comprises:

wherein R¹² is independently hydroxy, alkoxy, or comprises the half-lifeextending molecule; and cis 1 or
 2. 13. The mTA of claim 1, wherein thestaple covalently attached to the half-life extending molecule isselected from the group consisting of: